U.S. patent application number 09/901812 was filed with the patent office on 2002-11-21 for methods for enhancing the efficacy of cancer therapy.
Invention is credited to Pennica, Diane, Polakis, Paul, Szeto, Wayne, Tice, David.
Application Number | 20020173461 09/901812 |
Document ID | / |
Family ID | 27390600 |
Filed Date | 2002-11-21 |
United States Patent
Application |
20020173461 |
Kind Code |
A1 |
Pennica, Diane ; et
al. |
November 21, 2002 |
Methods for enhancing the efficacy of cancer therapy
Abstract
The invention concerns the identification of tumor antigens the
expression of which is selectively upregulated by retinoid
treatment. The invention further concerns improved methods of
cancer treatment and, in particular, methods enhancing the efficacy
of the treatment of cancers characterized by aberrant Wnt signaling
by administration of retinoic acid or other retinoids.
Inventors: |
Pennica, Diane; (Burlingame,
CA) ; Polakis, Paul; (Burlingame, CA) ; Szeto,
Wayne; (San Francisco, CA) ; Tice, David; (San
Mateo, CA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
620 NEWPORT CENTER DRIVE
SIXTEENTH FLOOR
NEWPORT BEACH
CA
92660
US
|
Family ID: |
27390600 |
Appl. No.: |
09/901812 |
Filed: |
July 10, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60228914 |
Aug 29, 2000 |
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60175849 |
Jan 13, 2000 |
|
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60197089 |
Apr 14, 2000 |
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Current U.S.
Class: |
424/156.1 ;
514/19.4; 514/19.5; 514/559; 514/725 |
Current CPC
Class: |
A61K 38/1709 20130101;
A61P 31/00 20180101; A61K 2039/505 20130101; C12N 15/85 20130101;
C12N 15/1034 20130101; A61P 35/00 20180101; G01N 33/574 20130101;
C07K 14/47 20130101; C07K 2319/00 20130101; A61K 38/1709 20130101;
A61K 2300/00 20130101 |
Class at
Publication: |
514/12 ; 514/559;
514/725 |
International
Class: |
A61K 038/17; A61K
031/203; A61K 031/07 |
Claims
What is claimed is:
1. A method for the selective enhancement of the expression of a
protein in a tumor cell characterized by aberrant Wnt signaling
comprising treating said tumor cell with an effective amount of a
retinoid.
2. The method of claim 1 wherein said protein is characterized by
synergistic enhancement of its expression by a combination of Wnt-1
and said retinoid.
3. The method of claim 2 wherein said protein is a cell surface
protein.
4. The method of claim 2 wherein said protein is over-expressed in
tumor cells relative to corresponding normal cells.
5. The method of claim 2 wherein said protein is selected from the
group consisting of 4-1BB ligand, ephrin b1, ISLR, autotaxin, and
Stra6.
6. The method of claim 5 wherein said protein is selected from the
group consisting of 4-1BB ligand, ephrin b1, ISLR, and Stra 6.
7. The method of claim 6 wherein said protein is selected from the
group consisting of 4-1BB ligand, ephrin b1, and ISLR.
8. The method of claim 1 wherein said retinoid is a retinoic
acid.
9. The method of claim 1 wherein said tumor is a human cancer.
10. The method of claim 9 wherein said human cancer is selected
from the group consisting of ovarian cancer, endometrial cancer,
Wilm's kidney tumor, colon cancer, breast cancer, prostate cancer,
gastric cancer, lung cancer, hepatocellular cancer, and
melanoma.
11. A method for the treatment of a tumor characterized by aberrant
Wnt signaling comprising treating said tumor with an effective
amount of a combination of a retinoid and an anti-tumor agent,
wherein said anti-tumor agent targets a protein in said tumor the
expression of which is enhanced by retinoid treatment.
12. The method of claim 11 wherein the expression of said protein
is synergistically enhanced by a combination of Wnt-1 and a
retinoid.
13. The method of claim 11 wherein said protein is a cell surface
protein.
14. The method of claim 11 wherein said protein is selected from
the group consisting of 4-1BB ligand, ephrin b1, ISLR, autotaxin
and Stra6.
15. The method of claim 14 wherein said protein is selected from
the group consisting of 4-1BB ligand, ephrin b1, ISLR and Stra
6.
16. The method of claim 15 wherein said protein is selected from
the group consisting of 4-1BB ligand, ephrin b1 and ISLR.
17. The method of claim 11 wherein said retinoid is administered
prior to the administration of said anti-tumor agent.
18. The method of claim 11 wherein said retinoid is administered
concurrently with the administration of said anti-tumor agent.
19. The method of claim 11 wherein said retinoid is administered
following the administration of said anti-tumor agent.
20. The method of claim 11 wherein said anti-tumor agent is an
antibody.
21. The method of claim 20 wherein said antibody is an antibody
fragment.
22. The method of claim 21 wherein said antibody fragment is
selected from the group consisting of Fab, Fab', F(ab').sub.2, and
Fv fragments, diabodies, single-chain antibody molecules, and
multispecific antibodies formed from antibody fragments.
23. The method of claim 20 wherein said antibody is a chimeric
antibody.
24. The method of claim 20 wherein said antibody is a humanized
antibody.
25. The method of claim 20 wherein said antibody is a human
antibody.
26. The method of claim 20 wherein said antibody is conjugated to a
cytotoxic agent.
27. The method of claim 26 wherein said cytotoxic agent is a
toxin.
28. The method of claim 27 wherein said toxin is a
maytansinoid.
29. The method of claim 20 wherein said antibody is produced in CHO
cells.
30. The method of claim 20 wherein said antibody is produced in
bacteria.
31. The method of claim 20, further comprising treatment with a
chemotherapeutic agent.
32. The method of claim 20, further comprising radiation
treatment.
33. The method of claim 11 wherein said tumor is a human
cancer.
34. The method of claim 33 wherein said human cancer is selected
from the group consisting of ovarian cancer, endometrial cancer,
Wilm's kidney tumor, colon cancer, breast cancer, prostate cancer,
gastric cancer, lung cancer, hepatocellular cancer, and
melanoma.
35. A method for identifying a gene target for tumor treatment
comprising: (a) contacting a cell expressing a Wnt proto-oncogene
with a retinoid; (b) determining the gene expression profile of
said cell; and (c) identifying a gene the expression of which is
enhanced by said retinoid treatment relative to its expression in a
corresponding untreated cell, as a target for tumor treatment.
36. The method of claim 35 wherein said cell is engineered to
conditionally express said Wnt proto-oncogen.
37. The method of claim 36 wherein said proto-oncogen is Wnt-1.
38. The method of claim 36 further comprising the step of inducing
the expression of said Wnt-1 and identifying a gene the expression
of which is synergistically enhanced by said tumor treatment and
Wnt-1 signaling, as a target for tumor treatment.
39. The method of claim 35 wherein said cell is a tumor cell.
40. The method of claim 35 comprising identifying a gene the
expression of which is selectively enhanced by said retinoid
treatment relative to a normal cell treated with said retinoid, as
a target for tumor treatment.
41. The method of claim 39 wherein said tumor cell is from a frozen
tumor sample.
42. The method of claim 41 wherein said tumor cell is from a
paraffin-embedded, formalin-fixed tumor sample.
43. The method of claim 35 wherein the gene expression profile is
determined by reverse transcriptase-PCR (RT-PCR) analysis.
44. The method of claim 35 wherein the gene expression profile is
determined by in situ hybridization.
45. The method of claim 35 wherein the gene expression profile is
determined by northern blotting.
46. A method for the treatment of a tumor in a mammalian subject
comprising the steps of: (a) incubating a sample of said tumor with
a retinoid; (b) determining the gene expression profile of said
sample prior to and following said incubation; (c) identifying a
gene the expression of which is enhanced by said retionid; and (d)
treating said patient with a combination of a retinoid and an
anti-tumor agent targeting said gene.
47. The method of claim 46 wherein said sample is additionally
incubated with Wnt-1.
48. The method of claim 47 comprising identifying, in step (c) a
gene the expression of which is synergistically enhanced by a
combination of said retinoid and Wnt-1.
49. The method of claim 46 wherein said anti-tumor agent is an
antibody.
50. The method of claim 49 wherein said antibody is an antibody
fragment.
51. The method of claim 50 wherein said antibody fragment is
selected from the group consisting of Fab, Fab', F(ab').sub.2, and
Fv fragments, diabodies, single-chain antibody molecules, and
multispecific antibodies formed from antibody fragments.
52. The method of claim 49 wherein said antibody is a chimeric
antibody.
53. The method of claim 49 wherein said antibody is a humanized
antibody.
54. The method of claim 49 wherein said antibody is a human
antibody.
55. The method of claim 49 wherein said antibody is conjugated to a
cytotoxic agent.
56. The method of claim 55 wherein said cytotoxic agent is a
toxin.
57. The method of claim 56 wherein said toxin is a
maytansinoid.
58. The method of claim 49 wherein said antibody is produced in CHO
cells.
59. The method of claim 50 wherein said antibody is produced in
bacteria.
60. The method of claim 46 further comprising treatment with a
chemotherapeutic agent.
61. The method of claim 46 further comprising radiation
treatment.
62. A method for diagnosing a cancer characterized by aberrant Wnt
signaling in a mammalian subject comprising (a) contacting a
biological sample obtained from said patient with retinoic acid;
(b) detecting the gene expression profile is said biological
sample; and (c) detecting a tumor antigen the expression of which
is enhanced by said retinoid treatment.
63. The method of claim 62 wherein said tumor antigen is selected
from the group consisting of 4-1BB ligand, ephrin b1, ESLR,
autotaxin, and Stra6.
64. An article of manufacture comprising: a container; an
anti-tumor agent within said container; and instructions to
administer said anti-tumor agent in combination with a
retinoid.
65. The article of manufacture of claim 64 further comprising a
retinoid.
66. The article of manufacture of claim 65 wherein said retinoid is
a retinoic acid.
Description
[0001] This is a non-provisional application which claims priority
under 35 U.S.C. .sctn.119(e) to provisional application No.
60/228,914 filed on Aug. 29, 2000, and under 35 U.S.C. .sctn.120 of
copending U.S. application Ser. No. 09/759,056 filed on Jan. 21,
2001, which in turn claims priority to provisional application Nos.
60/175,849 filed on Jan. 13, 2000, 60/197,089 filed on Apr. 14,
2000, and 60/228,914 filed on Aug. 29, 2000, the disclosures of
which are hereby expressly incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention concerns the identification of drug
targets for cancer therapy. In particular, the invention concerns
the identification of tumor antigens the expression of which is
selectively enhanced by retinoid treatment, and treatment methods
targeting such antigens.
[0004] 2. Description of the Related Art
[0005] One approach to understanding the molecular basis of cancer
and to devise effective treatment strategies is to identify
differences in gene expression between cancer cells and normal
cells. Strategies based on assumptions that steady-state mRNA
levels will differ between normal and malignant cells have been
used to clone differentially expressed genes (Zhang et al., Science
276:1268-1272 (1997)), and have lead to the identification of novel
drug targets.
[0006] The aberrant growth and survival of neoplastically
transformed cells is attributed to underlying genetic defects that
alter normal cellular homeostasis. Wnt signaling represents a
mechanism that contributes to the progression in a high percentage
of human cancers for which appropriate animal and cell culture
models are available. .beta.-catenin, a key component of the Wnt
signaling pathway, interacts with the TCF/LEF family of
transcription factors and activates transcription of Wnt target
genes. Recent studies have revealed that a number of proteins such
as, the adenomatous polyposis (APC) tumor suppressor, and axin are
involved in the regulation of the Wnt signaling pathway.
Furthermore, mutations in APC or .beta.-catenin have been found to
be responsible for the genesis of many human cancers.
[0007] For example, in the case of colorectal cancer, inactivation
of the APC tumor suppressor occurs early in tumor progression and
provides a growth advantage resulting from the inappropriate
activation of genes such as cyclin D and c-myc (He et al., Science
281:1509-1512 (1998)); Tetsu and McCormick, Nature 38:422-426
(1999)). These genes are targets of LEF/TCF transcription factors
that are activated by their interaction with .beta.-catenin, a
protein that is normally down-regulated by APC (Barker et al., Adv
Cancer Res 77:1-24 (2000); Polakis, Genes Dev 14:1837-1851 (2000)).
The upregulation of this signaling pathway in cancer can also
result from missense mutations in the .beta.-catenin gene
(Rubinfeld et al., Science 275:1790-1792 (1997)); Korinek et al.,
Science 275:1784-1787 (1997)). These mutations render the
.beta.-catenin protein refractory to down-regulation by APC.
Mutations in .beta.-catenin have recently been identified in a wide
variety of human tumors and are particularly prevalent in human
hepatocellular cancers (Polakis, 2000, supra). Activation of a
.beta.-catenin signaling also occurs when the cell surface frizzled
receptors are stimulated by the secreted Wnt ligands (Wodarz and
Nusse, Annu Rev Cell Dev Biol 14:59-88 (1998)). Although it is not
known whether Wnt ligands per se contribute to human cancers, early
experiments demonstrated that their over-expression in murine
mammary tissue was tumorigenic (Nusse and Varmus, Cell 31:99-109
(1982)). The Wnt signaling pathway has been implicated in the
pathogenesis of a variety of cancers, including colon cancer,
breast cancer, gastric cancer, lung cancer, and melanoma. Thus, the
vast majority of colorectal tumors contain mutations in the genes
coding for either the APC tumor suppressor or .beta.-catenin
(Polakis, Curr. Opin. Genet. Dev. 9:15-21 (1999)). Activating
mutations in .beta.-catenin have also been identified in cancers of
the ovary and endometrium, Wilm's kidney tumors and melanomas,
demonstrating that defects in Wnt-1 signaling contribute to the
progression of these cancers (Kobayashi et al., Jpn J Cancer Res
90:55-9 (1999); Koesters et al., Cancer Res 59:3880-2 (1999);
Palacios and Hamallo, Cancer Res 58:1344-7 (1998); Rimm et al., Am
J Pathol 154:325-9 (1999); Rubinfeld et al., Science 262:1731-1734
(1993); Wright et al., Int J Cancer 82:625-9 (1999)).
[0008] Signals emanating from the Wnt receptors are thought to
proceed via the activation of disheveled, which in turn, negatively
regulates glycogen synthase kinase 3b (GSK3b) (Peifer and Polakis,
Science 287:1606-1609 (2000)). This kinase normally phosphorylates
the regulatory sequence of .beta.-catenin that targets the protein
for ubiquitin-dependent degradation (Miller and Moon, Genes &
Dev. 10:2527-2539 (1996)). Negative regulation of GSK3b thus
increases the stability of .beta.-catenin and prolongs its
activation of the TCF/LEF transcription factors (Molenaar, et al.,
Cell 86:391-399 (1996); Behrens, et al., Nature 382:638-642
(1996)). Although the activation of the TCF/LEF transcription
factors by .beta.-catenin is well established, there remain
additional mechanisms independent of these transcription factors by
which .beta.-catenin might engage gene activation. One of these
alternative mechanisms was recently proposed by Byers and
colleagues in a study investigating the potential for cross talk
between signaling by retinoic acid receptors (RAR) and
.beta.-catenin (Easwaran, et al., Curr Biol 9:1415-1418 (1999)). A
synthetic reporter gene containing a retinoic acid response element
was activated more robustly by retinoids following the
over-expression of .beta.-catenin in a cultured cell line.
[0009] The use of monoclonal antibodies as therapeutics has gained
increased acceptance with several monoclonal antibodies (mAbs)
either approved for human use or in late stage clinical trials. The
first mAb approved by the US Food and Drug Administration (FDA) for
the treatment of allograft rejection was anti-CD3 (OKT3) in 1986.
Since then the pace of progress in the field of mAbs has been
considerably accelerated, particularly from 1994 onwards which led
to approval of additional seven mAbs for human treatment. These
include ReoPro.RTM. for the management of complications of coronary
angioplasty in 1994, Zenapax.RTM. (anti-CD25) for the prevention of
allograft rejection in 1997, Rituxan.RTM. (anti-CD20) for the
treatment of B cell non-Hodgkin's lymphoma in 1997, Infliximab.RTM.
(anti-TNF-.alpha.) initially for the treatment of Crohn's disease
in 1998 and subsequently for the treatment of rheumatoid arthritis
in 1999, Simulect.RTM. (anti-CD25) for the prevention of allograft
rejection in 1998, Synagis.RTM. (anti-F protein of respiratory
syncitial virus) for the treatment of respiratory infections in
1998, and Herceptin.RTM. (anti-HER2/neu) for the treatment of HER2
overexpressing metastatic breast tumors in 1998 (Glennie and
Johnson, Immunol Today 21: 403-410 (2000)).
[0010] The proven utility of therapeutic antibodies in the
treatment of human cancer in the clinic has recently spurned
intense activity aimed at the development and refinement of
immunotherapeutics. Ideally, these therapies require the presence
of cell surface antigens expressed on the cancer cells at
significantly higher levels than that present on normal tissues
throughout the body. Such criteria for differential expression on
tumors relative to normal tissue will obviously limit the number of
antigens considered desirable as targets for cancer treatment, such
as immunotherapy. Therefore, it would be desirable to find ways of
selectively enhancing cell surface antigen expression on tumor
cells. In particular, reagents that would selectively enhance the
level of antigen expression of cancer cells relative to normal
cells would have great potential in improving the therapeutic index
for treatment, such as immunotherapeutics directed against these
antigens.
SUMMARY OF THE INVENTION
[0011] Novel drug targets can be identified by differential
analysis of RNA transcripts isolated from cancer cell lines and
tissues. In the work underlying the present invention, this
approach has been extended by analyzing differences in gene
expression resulting from the drug treatment of transformed and
non-transformed cancer cells. A breast epithelial cell line, which
conditionally expresses the Wnt-1 proto-oncogen, was left untreated
or treated with 9-cis retinoic acid in the presence of absence of
Wnt-1 expression. A number of genes, including several cell surface
antigens were selectively up-regulated by the combination of Wnt-1
and retinoic acid. This observation indicates that the efficacy of
cancer treatment, and in particular, the immnunotherapy of cancers
characterized by the involvement of the Wnt signaling pathway can
be enhanced by co-administration of retinoic acid or other
retinoids.
[0012] Accordingly, in one aspect the invention concerns a method
for the selective enhancement of the expression of a protein in a
tumor cell characterized by aberrant Wnt signaling comprising
treating the tumor cell with an effective amount of a retinoid.
[0013] In a particular embodiment, the protein is characterized by
synergistic enhancement of its expression by a combination of Wnt-1
and retinoid treatment.
[0014] In a preferred embodiment, the protein is a cell surface
protein, which is over-expressed in tumor relative to corresponding
normal cells. Exemplary proteins include, for example, protein
4-1BB ligand, ephrin b1, ISLR, autotaxin, and Stra6.
[0015] The treatment can be performed by any retinoid, including
retinoic acid.
[0016] In a particular embodiment, the tumor is a human cancer,
such as, for example, ovarian cancer, endometrial cancer, Wilm's
kidney tumor, colon cancer, breast cancer, prostate cancer, gastric
cancer, lung cancer, hepatocellular cancer, or melanoma.
[0017] In another aspect, the invention concerns a method for the
treatment of a tumor characterized by aberrant Wnt signaling
comprising treating the tumor with an effective amount of a
combination of a retinoid and an anti-tumor agent, wherein the
anti-tumor agent targets a protein in the tumor the expression of
which is synergistically enhanced by a combination of Wnt-1 and
retinoid treatment.
[0018] The targeted protein preferably is a cell surface protein,
such as, for example, 4-1BB ligand, ephrin b1, ISLR, autotaxin, or
Stra6. The retinoid may be administered prior to, concurrently
with, or following the administration of the anti-tumor agent.
[0019] In a preferred embodiment, the anti-tumor agent is an
antibody. Throughout the disclosure, the term "antibody" is used in
the broadest sense, and includes antibody fragments, such as Fab,
Fab', F(ab').sub.2, and Fv fragments, diabodies, single-chain
antibody molecules, and multispecific antibodies formed from
antibody fragments. The antibody may be a chimeric, humanized, or
human antibody, including antibody fragments.
[0020] The treatment may optionally include additional treatment,
for example with a chemotherapeutic agent and/or radiation
treatment.
[0021] The tumor preferably is a human cancer, such as, for example
ovarian cancer, endometrial cancer, Wilm's kidney tumor, colon
cancer, breast cancer, prostate cancer, gastric cancer, lung
cancer, hepatocellular cancer, and melanoma.
[0022] In a further aspect, the invention concerns a method for
identifying a target gene for tumor treatment comprising contacting
a tumor cell with a retinoid and identifying a gene which is
selectively upregulated by the retinoid. In particular, the target
gene is identified by
[0023] (a) contacting a ell expressing a Wnt proto-oncogene with a
retinoid;
[0024] (b) determining the gene expression profile of the cell;
and
[0025] (c) identifying a gene which is selectively upregulated by
the retinoid treatment relative to a corresponding untreated cell,
as a target for tumor treatment.
[0026] In a preferred embodiment, the tumor cell is additionally
contacted with Wnt-1, and a protein the expression of which is
synergistically enhanced by the retinoid and Wnt-1 is
identified.
[0027] In yet another aspect, the invention concerns a method for
the treatment of a tumor in a mammalian subject comprising the
steps of:
[0028] (a) incubating a sample of the tumor with a retinoid;
[0029] (b) determining the gene expression profile of the sample
prior to and following the incubation;
[0030] (c) identifying a gene the expression of which is enhanced
by the retinoid; and treating the patient with an anti-tumor agent
targeting the gene identified.
[0031] In a preferred embodiment, the sample is additionally
incubated with Wnt-1, and a gene is identified the expression of
which is synergistically enhanced by a combination of the retinoid
and Wnt-1.
[0032] Just as in the previous embodiment, the anti-tumor agent may
be an antibody, including antibody fragments, such as Fab, Fab',
F(ab').sub.2, and Fv fragments, diabodies, single-chain antibody
molecules, and multispecific antibodies formed from antibody
fragments. The antibodies or antibody fragments may be chimeric,
humanized, or human.
[0033] The treatment may further comprise the administration of a
chemotherapeutic agent, which may, for example, be a cytotoxic
molecule, optionally fused to an antibody specifically binding the
protein identified and/or radiation treatment.
[0034] In yet another aspect, the invention concerns method for the
diagnosis of cancer associated with aberrant Wnt signaling in a
mammalian patient based on the detection of a tumor antigen
upregulated by retinoid treatment. The biological sample may, for
example, be a tumor cell or tissue.
[0035] In a still further aspect, the invention concerns articles
of manufacture comprising an anti-tumor agent and instructions to
administer such anti-tumor agent in combination with a retinoid,
such as retinoic acid. The article of manufacture may additionally
comprise a retinoid.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] FIG. 1 shows the nucleotide sequence (SEQ ID NO: 1) of a
cDNA containing a nucleotide sequence (nucleotides 1-2732) encoding
native sequence PRO10282 (stra6), wherein the nucleotide sequence
(SEQ ID NO: 1) is a clone designated herein as "DNA148380-2827."
Also presented in bold font and underlined are the positions of the
respective start and stop codons.
[0037] FIG. 2 shows the amino acid sequence (SEQ ID NO: 2) of a
native sequence PRO10282 (stra6) polypeptide as derived from the
coding sequence of SEQ ID NO: 1. Also shown are the approximate
locations of various other important polypeptide domains.
[0038] FIGS. 3A-D show hypothetical exemplifications for using the
below described method to determine % amino acid sequence identity
(FIGS. 3A-B) and % nucleic acid sequence identity (FIGS. 3C-D)
using the ALIGN-2 sequence comparison computer program, wherein
"PRO" represents the amino acid sequence of a hypothetical PRO10282
polypeptide of interest, "Comparison Protein" represents the amino
acid sequence of a polypeptide against which the "PRO" polypeptide
of interest is being compared, "PRO-DNA" represents a hypothetical
PRO10282-encoding nucleic acid sequence of interest, "Comparison
DNA" represents the nucleotide sequence of a nucleic acid molecule
against which the "PRO-DNA" nucleic acid molecule of interest is
being compared, "X, "Y" and "Z" each represent different
hypothetical amino acid residues and "N", "L" and "V" each
represent different hypothetical nucleotides.
[0039] FIGS. 4A-Q provide the complete source code for the ALIGN-2
sequence comparison computer program. This source code may be
routinely compiled for use on a UNIX operating system to provide
the ALIGN-2 sequence comparison computer program.
[0040] FIG. 5 shows a nucleotide sequence designated herein as
DNA100038 (SEQ ID NO: 3).
[0041] FIG. 6 shows the nucleotide sequence (SEQ ID NO: 4) of a
cDNA containing a nucleotide sequence (nucleotides 1-2778) encoding
a native sequence human Stra6 polypeptide variant, wherein the
nucleotide sequence (SEQ ID NO: 4) is a clone designated herein as
"DNA148389-2827-1." Also presented in bold font and underlined are
the positions of the respective start and stop codons.
[0042] FIG. 7 shows the amino acid sequence (SEQ ID NO: 5) of a
native sequence human Stra6 polypeptide variant as derived from the
coding sequence of SEQ ID NO: 4. Also shown are the approximate
locations of various other important polypeptide domains.
[0043] FIG. 8 is a schematic representation of mouse Stra6, and the
human Stra6 protein encoded by DNA148380-2827 (native human
PRO10282).
[0044] FIG. 9 shows the hydrophobicity plot of the native sequence
human Stra6 protein encoded by DNA 148380-2827 (native human
PRO10282).
[0045] FIG. 10 shows the relative RNA expression profile for the
native sequence human Stra6 protein encoded by DNA 148380-2827 in
various normal human tissues.
[0046] FIG. 11 shows the RNA fold expression for the native
sequence human Stra6 protein encoded by DNA 148380-2827 in human
colon tumor tissue relative to RNA expression in normal mucose from
the same patient assayed by quantitative PCR. The data are from one
experiment done in triplicate. The experiment was repeated at least
twice with a different set of PCR primers.
[0047] FIG. 12A shows the RNA expression for the native sequence
human Stra6 protein encoded by DNA148380-2827 in human colon tissue
relative to RNA expression in normal mucose from the same patient,
using the housekeeping gene, GAPDH as a control.
[0048] FIG. 12B shows the localization of Stra6 to the epithelial
tumor cells in a colon adenocarcinoma by in situ hybridization.
[0049] FIG. 13 shows the RNA expression for the native human Stra6
protein encoded by DNA148380-2827 in human breast, kidney, colon
and lung tumor cell lines, relative to corresponding normal cell
lines.
[0050] FIG. 14 illustrates the expression of peptide fragments
derived from the native sequence human Stra6 protein encoded by
DNA148380-2827 in E. coli.
[0051] FIG. 15 illustrates Stra6 RNA expression in human colon
carcinoma cells in the presence and absence of all-trans-retinoic
acid (ATRA) and 9-cis-retinoic acid (9cRA), respectively.
[0052] FIG. 16 In situ hybridization for Stra6 in tumor sections.
Darkfield images demonstrating silver grains (A, C, E, G) are shown
with corresponding hematoxylin/eosin-stained brightfield images (B,
D, F, H). Moderate densities of silver grains overlie tumor cells
but not a blood vessel in a malignant melanoma (A, B). Neoplastic
epithelium in an endometrial adenocarcinoma is moderately labeled
whereas tumor stroma is negative (C, D). Blastemal regions in a
Wilm's tumor display high expression levels whereas tumor stroma is
negative (E, F). A pheochromocytoma shows very high Stra6 mRNA
expression while adjacent normal adrenal cortex is negative (G, H).
Scale bars=100 microns.
[0053] FIG. 17(A) Induction of Stra6 mRNA expression in response to
9-cis-RA or all-trans-RA in C57MG/Parent and C57MG/Wnt-1 cells. (B)
Induction of Stra6 mRNA expression in C57MG/Parent cells in
response to Wnt-3A conditioned media and 9-cis-RA. (C) Induction of
Stra6 mRNA expression after retinoic acid treatment in HCT116 and
WiDr colon adenocarcinoma cells. (D) Darkfield images demonstrating
Stra6 expression by in situ hybridization in HCT116 cells before
(top panel) and after (lower panel) treatment with retinoic acid.
(E) Stra6 protein expression in WiDr cells before (-RA) and after
(+RA) treatment with retinoic acid as visualized by Western blot
with a monoclonal antibody directed against human Stra6 peptide B.
(F) Stra6 membrane localization in WiDr cells untreated (left
panel) or treated (right panel) with retinoic acid.
Immunohistochemistry was performed with an anti-human Stra6 peptide
B monoclonal hybridoma culture supernatant (clone 12F4.2H9.1D5).
For the experiments shown in A-F, cells were treated with retinoic
acid for 48 hours. Stra6 products obtained after completion of the
quantitative PCR reactions (40 cycles each) are shown below each
graph A-C.
[0054] FIG. 18(A) Wnt-1 induces RAR.gamma.-1 expression.
Protein-equivalent amounts of whole cell lysate from tetracycline
repressible C57MG/Wnt-1 cells in the absence of tetracycline for 0,
24, 48, or 72 hours, were subjected to SDS-PAGE and immunoblotted
for RAR.gamma.-1 and ERK2. (B) RAR.gamma.-1 mRNA expression in
hyperplastic mammary glands and mammary gland tumors from Wnt-1
transgenic mice. mRNA expression was determined by quantitative
RT-PCR and the data are expressed as fold expression relative to
mRNA expression in wild-type mammary glands.
[0055] FIG. 19 illustrates the synergistic induction of stra6 by
retinoic acid (RA) and Wnt-1 (Wnt) treatment.
[0056] FIG. 20 illustrates the synergistic induction of autotaxin
by retinoic acid (RA and Wnt-1 (Wnt).
[0057] FIG. 21 illustrates the synergistic induction of the 41BB
ligand (41bb) by retinoic acid (RA) and Wnt-1 (Wnt).
[0058] FIG. 22 illustrates the synergistic induction of ephrinb 1
by retinoic aid (RA) and Wnt-1 (Wnt).
[0059] FIG. 23 shows that the ISRL gene is responsive to retinoic
acid (RA), but not to Wnt-1 (Wnt), while the combination of both
agents results in activation that exceeds that of retinoic acid
alone.
[0060] FIG. 24 shows that the tight junction protein ZO-1 and the
M-ras gene do not respond significantly either to retinoic acid
(RA) or Wnt-1 (Wnt) alone, but are activated by combined
treatment.
[0061] FIG. 25A illustrates the activation of an RAR response
element (RARE) by .beta.-catenin, as determined by the increased
production of luciferase.
[0062] FIG. 25B shows that the LEF responsive element TopFlash is
activated not only by .beta.-catenin, was further activated by
coexpression of LEF with .beta.-catenin.
[0063] FIG. 26 shows the upregulation of Stra6 mRNA in tumors and
normal mammary gland by various doses of all-trans-retinoic acid
(ATRA).
[0064] FIG. 27 shows the upregulation of Stra6 mRNA in WiDr
xenografts from mice orally dosed with 400 mg/kg of ATRA.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0065] A. Definitions
[0066] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. See,
e.g. Singleton et al., Dictionary of Microbiology and Molecular
Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994);
Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold
Springs Harbor Press (Cold Springs Harbor, N.Y. 1989). For purposes
of the present invention, the following terms are defined
below.
[0067] The terms "PRO10282 polypeptide", "PRO10282 protein",
"PRO10282", "Stra6 polypeptide", "Stra6 protein" and "Stra6" are
used interchangeably, and encompass native sequence PRO10282
(Stra6) and PRO10282 (Stra6) polypeptide variants (which are
further defined herein). The PRO10282 (Stra6) polypeptide may be
isolated from a variety of sources, such as from human tissue types
or from another source, or prepared by recombinant and/or synthetic
methods.
[0068] A "native sequence PRO10282" or "native sequence Stra6"
comprises a polypeptide having the same amino acid sequence as a
PRO10282 derived from nature. Such native sequence PRO10282 (Stra6)
can be isolated from nature or can be produced by recombinant
and/or synthetic means. The term "native sequence PRO10282" or
"native sequence Stra6" specifically encompasses
naturally-occurring truncated or secreted forms (e.g., an
extracellular domain sequence), naturally-occurring variant forms
(e.g., alternatively spliced forms) and naturally-occurring allelic
variants of the PRO10282. In one embodiment of the invention, the
native sequence PRO10282 is a mature or full-length native sequence
PRO10282 comprising amino acids 1 to 667 of FIG. 2 (SEQ ID NO: 2).
In another embodiment of the invention, the native sequence
PRO10282 polypeptide is a mature or full-length PRO19578
polypeptide comprising amino acids 1 to 658 of SEQ ID NO: 5, which
is believed to be an alternatively spliced form of the native
sequence PRO10282 polypeptide of SEQ ID NO: 2. Also, while the
PRO10282 polypeptides disclosed in FIG. 2 (SEQ ID NO: 2) and in
FIG. 7, SEQ ID NO: 5 are shown to begin with the methionine residue
designated herein as amino acid position 1, it is conceivable and
possible that another methionine residue located either upstream or
downstream from amino acid position 1 in FIG. 2 (SEQ ID NO: 2) or
in FIG. 7 (SEQ ID NO: 5) may be employed as the starting amino acid
residue for the PRO10282 polypeptide. All Stra6 molecules which
might be alternatively spliced at their 5' end are specifically
included within the definition herein.
[0069] The PRO10282 (Stra6) polypeptide "extracellular domain" or
"ECD" refers to a form of the PRO10282 (Stra6) polypeptide which is
essentially free of the transmembrane and cytoplasmic domains.
Ordinarily, a PRO10282 polypeptide ECD will have less than about 1%
of such transmembrane and/or cytoplasmic domains and preferably,
will have less than about 0.5% of such domains. It will be
understood that any transmembrane domain(s) identified for the
PRO10282 polypeptides of the present invention are identified
pursuant to criteria routinely employed in the art for identifying
that type of hydrophobic domain. The exact boundaries of a
transmembrane domain may vary but most likely by no more than about
5 amino acids at either end of the domain as initially identified.
As such, in one embodiment of the present invention, the
extracellular domain of a PRO10282 polypeptide comprises amino
acids 1 to X, wherein X is any amino acid from amino acid 49 to 59
of FIG. 2 (SEQ ID NO: 2) or of FIG. 7 (SEQ ID NO: 5).
[0070] "PRO10282 variant polypeptide" or "Stra6 variant
polypeptide", which terms are used interchangeably, means an active
PRO10282 (Stra6) polypeptide as defined below having at least about
80% amino acid sequence identity with the amino acid sequence of
(a) residues 1 to 667 of the PRO10282 polypeptide shown in FIG. 2
(SEQ ID NO: 2), or residues 1 to 658 of FIG. 7 (SEQ ID NO: 5), (b)
1 to X of FIG. 2 (SEQ ID NO: 2) or FIG. 7 (SEQ ID NO: 5), wherein X
is any amino acid from amino acid 49 to amino acid 59 of FIG. 2
(SEQ ID NO: 2) or of FIG. 7 (SEQ ID NO: 5), or (c) another
specifically derived fragment of the amino acid sequence shown in
FIG. 2 (SEQ ID NO: 2), or FIG. 7 (SEQ ID NO: 5). Such PRO10282
(Stra6) variant polypeptides include, for instance, PRO10282
(Stra6) polypeptides wherein one or more amino acid residues are
added, or deleted, at the N-and/or C-terminus, as well as within
one or more internal domains, of the sequence of FIG. 2 (SEQ ID NO:
2) or FIG. 7 (SEQ ID NO: 5). Ordinarily, a PRO10282 variant
polypeptide will have at least about 80% amino acid sequence
identity, more preferably at least about 81% amino acid sequence
identity, more preferably at least about 82% amino acid sequence
identity, more preferably at least about 83% amino acid sequence
identity, more preferably at least about 84% amino acid sequence
identity, more preferably at least about 85% amino acid sequence
identity, more preferably at least about 86% amino acid sequence
identity, more preferably at least about 87% amino acid sequence
identity, more preferably at least about 88% amino acid sequence
identity, more preferably at least about 89% amino acid sequence
identity, more preferably at least about 90% amino acid sequence
identity, more preferably at least about 91% amino acid sequence
identity, more preferably at least about 92% amino acid sequence
identity, more preferably at least about 93% amino acid sequence
identity, more preferably at least about 94% amino acid sequence
identity, more preferably at least about 95% amino acid sequence
identity, more preferably at least about 96% amino acid sequence
identity, more preferably at least about 97% amino acid sequence
identity, more preferably at least about 98% amino acid sequence
identity and yet more preferably at least about 99% amino acid
sequence identity with (a) residues 1 to 667 of the PRO10282
polypeptide shown in FIG. 2 (SEQ ID NO: 2) or residues 1 to 658 of
the PRO19578 polypeptide of FIG. 7 (SEQ ID NO: 5), (b) 1 to X of
FIG. 2 (SEQ ID NO: 2) or FIG. 7 (SEQ ID NO: 5), wherein X is any
amino acid from amino acid 49 to amino acid 59 of FIG. 2 (SEQ ID
NO: 2) or FIG. 7 (SEQ ID NO: 5), or (c) another specifically
derived fragment of the amino acid sequence shown in FIG. 2 (SEQ ID
NO: 2) or FIG. 7 (SEQ ID NO: 5). PRO10282 variant polypeptides do
not encompass the native PRO10282 polypeptide sequence. Ordinarily,
PRO10282 variant polypeptides are at least about 10 amino acids in
length, often at least about 20 amino acids in length, more often
at least about 30 amino acids in length, more often at least about
40 amino acids in length, more often at least about 50 amino acids
in length, more often at least about 60 amino acids in length, more
often at least about 70 amino acids in length, more often at least
about 80 amino acids in length, more often at least about 90 amino
acids in length, more often at least about 100 amino acids in
length, more often at least about 150 amino acids in length, more
often at least about 200 amino acids in length, more often at least
about 250 amino acids in length, more often at least about 300
amino acids in length, or more, and are different from fragments of
the murine Stra6 sequence, as disclosed in Bouillet et al.,
Mechanisms of Development 63, 173-186 (1997). Variants of other
polypeptides disclosed herein are defined in an analogous
manner.
[0071] "Percent (%) amino acid sequence identity" is defined as the
percentage of amino acid residues in a candidate sequence that are
identical with the amino acid residues in a reference (e.g. native
polypeptide) sequence, after aligning the sequences and introducing
gaps, if necessary, to achieve the maximum percent sequence
identity, and not considering any conservative substitutions as
part of the sequence identity. Alignment for purposes of
determining percent amino acid sequence identity can be achieved in
various ways that are within the skill in the art, for instance,
using publicly available computer software such as BLAST, BLAST-2,
ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the
art can determine appropriate parameters for measuring alignment,
including any algorithms needed to achieve maximal alignment over
the full-length of the sequences being compared. For purposes
herein, however, % amino acid sequence identity values are obtained
as described below by using the sequence comparison computer
program ALIGN-2, wherein the complete source code for the ALIGN-2
program is provided in FIGS. 4A-Q. The ALIGN-2 sequence comparison
computer program was authored by Genentech, Inc. and the source
code shown in FIGS. 4A-Q has been filed with user documentation in
the U.S. Copyright Office, Washington D.C., 20559, where it is
registered under U.S. Copyright Registration No. TXU510087. The
ALIGN-2 program is publicly available through Genentech, Inc.,
South San Francisco, Calif. or may be compiled from the source code
provided in FIGS. 4A-Q. The ALIGN-2 program should be compiled for
use on a UNIX operating system, preferably digital UNIX V4.0D. All
sequence comparison parameters are set by the ALIGN-2 program and
do not vary.
[0072] For purposes herein, the % amino acid sequence identity of a
given amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows:
100 times the fraction {fraction (X/Y)}
[0073] where X is the number of amino acid residues scored as
identical matches by the sequence alignment program ALIGN-2 in that
program's alignment of A and B, and where Y is the total number of
amino acid residues in B. It will be appreciated that where the
length of amino acid sequence A is not equal to the length of amino
acid sequence B, the % amino acid sequence identity of A to B will
not equal the % amino acid sequence identity of B to A. As examples
of % amino acid sequence identity calculations, FIGS. 3A-B
demonstrate how to calculate the % amino acid sequence identity of
the amino acid sequence designated "Comparison Protein" to the
amino acid sequence designated "PRO".
[0074] Unless specifically stated otherwise, all % amino acid
sequence identity values used herein are obtained as described
above using the ALIGN-2 sequence comparison computer program.
However, % amino acid sequence identity may also be determined
using the sequence comparison program NCBI-BLAST2 (Altschul et al.,
Nucleic Acids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence
comparison program may be downloaded from
http://www.ncbi.nlm.nih.gov. NCBI-BLAST2 uses several search
parameters, wherein all of those search parameters are set to
default values including, for example, unmask=yes, strand=all,
expected occurrences=10, minimum low complexity length=15/5,
multi-pass e-value=0.01, constant for multi-pass=25, dropoff for
final gapped alignment=25 and scoring matrix=BLOSUM62.
[0075] In situations where NCBI-BLAST2 is employed for amino acid
sequence comparisons, the % amino acid sequence identity of a given
amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows:
100 times the fraction {fraction (X/Y)}
[0076] where X is the number of amino acid residues scored as
identical matches by the sequence alignment program NCBI-BLAST2 in
that program's alignment of A and B, and where Y is the total
number of amino acid residues in B. It will be appreciated that
where the length of amino acid sequence A is not equal to the
length of amino acid sequence B, the % amino acid sequence identity
of A to B will not equal the % amino acid sequence identity of B to
A.
[0077] The terms "4-1BB ligand" and "4-1BBL" are used
interchangeably and refer to a native sequence membrane protein
belonging to the tumor necrosis factor superfamily, typically
expressed on antigen presenting cells, and its variants. The
nucleic acid and deduced amino acid sequences of a native human
4-1BB ligand are disclosed in Zhou et al., Immunol Lett 45:67-73
(1995). The amino acid sequence variants of native sequence 4-1BB
ligands are defined on the analogy of the Stra6 protein discussed
above, using the same algorithm to determine sequence identity. It
has been shown that antibodies to the receptor of 4-1BBL (4-1BB)
can eradicate established tumors in mice, and the 4-1BB T cell
stimulation pathway has been implicated in the amplification of an
antitumor immune response.
[0078] The term "ephrin b1" is used herein to describe native
sequence ephrin b1 molecules (also known as "lerk2") and their
variants, which are defined as described above for Stra6. Human
ephrin b1 is a 45 kDa, 346 amino acid glycosylated polypeptide that
contains a 24 amino acid signal sequence, a 211 amino acid
extracellular region, a 26 amino acid transmembrane (TM) region,
and an 83 amino acid cytoplasmic segment (Davis, et al., Science
266:816 (1994); Beckmann, et al., EMBO J 13:3657 (1994)). There is
an about 95% amino acid sequence identity between the extracellular
regions of the mouse and human ephrin b1 polypeptides. A potential
proteolytic cleavage site on ephrin b1 has been identified
(Beckmann et al., supra). The role of ephrins in angiogenesis and
in tumor progression is discussed, for example, in L'Allemain et
al., Bull Cancer 87:529-530 (2000).
[0079] The terms "ISLR," "immunoglobulin superfamily containing
leucine rich repeat," and "immunoglobulin superfamily containing
LRR" are used interchangeably, and refer to native sequence ISRL
proteins and their variants, as hereinabove defined. The cloning
and sequencing of a native human ISLR was reported by Nagasawa et
al., Genomics 44:173-179 (1997). Further human and mouse ISLR genes
are described in Nagasawa et al., Genomics 61:37-43 (1999).
[0080] The terms "autotaxin" and "ATX" are used interchangeably,
and refer to native sequence autotaxin molecules of any animal
species, and autotaxin variants, as hereinabove defined. Autotaxin
is a cancer-related autocrine motility factor, which shows
significant homology to the plasma cell membrane differentiation
antigen PC-1. Human autotaxin is a 125-kDa, 915 aa glycoprotein
originally isolated from a human melanoma cell line (Murata et al.,
J Biol Chem 269:30479-84 (1994)). The cloning of autotaxin from
human teratoarcinoma cells has been reported by Lee et al., Biochem
Biophys Res Commun 218:714-719 (1996). Autotoxin has been described
to catalyze the hydrolysis of the phosphodiester bond on either
side of the beta-phosphate of ATP, and also to catalyze the
hydrolysis of GTP to GDP and GMP, of either AMP or PPi to Pi, and
the hydrolysis of NAD to AMP. Each of these substrates can serve as
a phosphate donor in the phosphorylation of autotoxin. Autotoxin is
believed to be a potent tumor motogen, which augments invasive and
metastatic potential of ras-transformed cells (Nam et al., Oncogene
19:241-247 (2000)).
[0081] The term "retinoid" is used in the broadest sense and
specifically includes, without limitation, retinoic acid (also
known as tretinoin, vitamin A acid or vitamin A.sub.1), and
retinoic acid derivatives consisting of four isoprenoid units
joined in a head-to-tail manner, such as retinol, retinal,
substituted retinoids, seco-, nor-, and retro-retinoids. All
retinoids may be formally derived from a monocyclic parent compound
containing five carbon-carbon double bonds and a functional group
at the terminus of the acyclic portion. For the nomenclature of
retinoids see Moss, G. P., Arch. Biochem. Biophys., 224:728-731
(1983); Eur. J. Biochem., 129:1-5 (1982); J. Biol. Chem.,
258:5329-5333 (1983); Pure Appl. Chem., 55:721-726 (1983);
Biochemical Nomenclature and Related Documents, 2.sup.nd edition,
Portland Press, 1992, pages 247-251. The term "retinoid"
specifically includes molecules occurring in nature and their
derivatives, as long as they retain retinoid biological
activity.
[0082] The term "retinoid biological activity," as defined herein,
refers to involvement in embryogenesis, (epithelial) cell
proliferation, cell differentiation and/or carcinogenesis,
especially oncogenic effect associated with cancers characterized
by aberrant Wnt signaling.
[0083] The term "Wnt" refers to a family of highly conserved,
cysteine-rich, secreted glycoproteins that are involved in critical
aspects of early embryonic development. Wnt genes are also
implicated in cancer. "Wnt" as used herein specifically includes
Wnt genes of all human and non-human animal species, including, but
not limited to, mammals, such as human, mouse, rat and other
rodents, etc. The term "Wnt" specifically includes native human Wnt
genes and encoded polypeptides, including human Wnt-1 (previously
called int-1) (van Ooyen et al., EMBO J 4:2905-9 (1985)); Wnt-2
(previously called Dint-1) (Wainwright et al., EMBO J 7:1743-1748
(1988)); Wnt-13 (Katoh et al., Oncogene 13:873-876 (1996)); Wnt-3
(Roelink et al., Genomics 17:790-792 (1993)); Huguet et al., Cancer
Res 54:2615-2521 (1994)); Wnt-4 (Huguet et al., 1994, supra);
Wnt-5A (Clark et al., Genomics 18:249-260 (1993)); Wnt-6 (Rankin et
al., Cytogenet Cell Genet 84:50-52 (1999)); Wnt-7A (Ikegawa et al.,
Cytogenet Cell Genet 74:149-152 (1996)); Wnt-7B (Huguet et al.,
1994, supra); Wnt-8B (Lako et al, Genomics 35:386-388 (1996));
Wnt-10B (Bui et al., Oncogene 14:1249-1253 (1997)); Wnt-11 (Lako et
al., Gene 219:101-110 (1998)); Wnt-14 (Bergstein et al., Genomics
46:450-458 (1997)); and Wnt-16 (McWhirter et al., Proc Natl Acad
Sci USA 96:11464-11469 (1999); Fear et al., Biochem Biophys Res
Commun 278:814-820 (2000)), and their variants, in particular amino
acid sequence variants, which have at least about 80%, more
preferably at least about 85%, more preferably at least about 90%,
even more preferably at least about 95%, yet more preferably at
least about 98%, more preferably at least about 99% percent amino
acid sequence identity with a native Wnt polypeptide, wherein
sequence identity is determined as hereinabove defined. In a
preferred embodiment, the term "Wnt" refers to any transforming
(oncogenic) Wnt polypeptide, as discussed in Wong et al., Mol Cell
Biol 14:6278-86 (1994).
[0084] A cancer is "characterized by aberrant Wnt signaling" if it
harbors genetic defects and/or shows altered expression patterns
(including mutations, amplification, over-expression and/or
suppression) of any member of a Wnt signaling pathway. Wnts exert
their effects through interaction with cell surface receptors,
named "Frizzled." Structurally, the Frizzled receptors have an
extracellular Wnt-binding domain, seven transmembrane-spanning
regions, and an intracellular C-terminal tail. Additional
components of the Frizzled receptor-Wnt interaction pathway are the
soluble Wnt inhibitors which consist of secreted proteins
containing a cysteine-rich domain (CRD) similar to that in the
ligand binding domain of the Frizzled receptors. These Wnt
inhibitory proteins are collectively referred to as Frizzled
Receptor-like Proteins (FRPs). Since the Frizzled receptors have no
enzymatic motifs on their intracellular domains, the Wnt signals
are believed to be transmitted through receptor coupling to
Dishevelled (dsh) proteins. The next step in the Wnt signaling
cascade is the inhibition of the serine/threonine kinase, glycogen
synthetase kinase-3b (GSK-3b) by Dishevelled. The consequence of
the Wnt-induced inhibition of GSK-3b activity is that the next
protein in the cascade, .beta.-catenin, is stabilized. The central
region of the .beta.-catenin protein contains a positively charged
groove which is believed to interact with acidic regions in the
adenometous polyposis coli (APC) locus encoded protein, the
transcription factor TCF/LEF1, and the cadherin family of
calcium-dependent cell-adhesion molecules. Members of the Wnt
signaling pathway, including APC and .beta.-catenin, have been
implicated in the pathogenesis of a series of human cancers,
including colon cancer, breast cancer, hepatocellular cancers, and
melanoma (Morin et al., Science 275:1787-1790 (1997)). Mutations in
specific regions of either gene can cause the stabilization and
accumulation of cytoplasmic .beta.-catenin, which is believed to
contribute to human carcinogenesis through the activation of target
genes such as the WISP genes (Pennica et al., Proc. Natl. Acad.
Sci. USA 95:14717-14722 (1998)). Mutations in the Wnt-1 gene are
also involved in the development of Wilm's tumors, a kidney cancer
found in children. As discussed above, further members of the Wnt
signaling pathway include, without limitation, other members of the
Wnt gene family, Frizzled receptors, the cytoplasmic protein
Dishevelled (Dsh), glycogen synthase kinase-3.beta. (GSK-3.beta.),
the transcription factor TCF/LEF1, and transcriptionally activated
downstream components of the pathway, such as the nodal-related 3
gene, Xnr3, the homeobox genes, engrailed, goosecoid, twin (Xtwn),
and siamois, c-myc, and the WISP genes, e.g. WISP-1 and WISP-2. The
term "characterized by aberrant Wnt signaling" includes genetic
defects and/or altered expression patterns (including mutations,
amplification, over-expression and/or suppression) of any of these
members of the Wnt signaling pathway, or any other members, known
today or hereinafter identified.
[0085] The enhancement of the expression of an antigen in a tumor,
e.g. cancer, is "selective," if the enhancement of the expression
level of such antigen in tumor cells is significantly higher than
in normal cells. Preferably, the enhancement of the expression
level of a target antigen (e.g. a cell surface antigen) in tumor
cells results in at least about two-fold, more preferably at least
about three-fold, even more preferably at least about five-fold,
most preferably at least about ten-fold over-expression in tumor
cells or tissues relative to corresponding normal (non-tumorigenic)
cells or tissues.
[0086] "Tumor", as used herein, refers to all neoplastic cell
growth and proliferation, whether malignant or benign, and all
pre-cancerous and cancerous cells and tissues.
[0087] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. Examples of cancer include but are not
limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
More particular examples of such cancers include breast cancer,
prostate cancer, colon cancer, squamous cell cancer, small-cell
lung cancer, non-small cell lung cancer, gastrointestinal cancer,
pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer,
liver cancer, bladder cancer, hepatoma, colorectal cancer,
endometrial carcinoma, salivary gland carcinoma, kidney cancer,
liver cancer, vulval cancer, thyroid cancer, hepatic carcinoma and
various types of head and neck cancer. Cancers particularly
amenable to treatment in accordance with the present invention
include ovarian cancer, endometrial cancer, Wilm's kidney tumor,
colon cancer, breast cancer, prostate cancer, gastric cancer, lung
cancer, hepatocellular cancer, and melanoma.
[0088] "Treatment" refers to both therapeutic treatment and
prophylactic or preventative measures, wherein the object is to
prevent or slow down (lessen) the targeted pathologic condition or
disorder. Those in need of treatment include those already with the
disorder as well as those prone to have the disorder or those in
whom the disorder is to be prevented. In tumor (e.g., cancer)
treatment, a therapeutic agent may directly decrease the pathology
of tumor cells, or render the tumor cells more susceptible to
treatment by other therapeutic agents, e.g., radiation and/or
chemotherapy.
[0089] The "pathology" of cancer includes all phenomena that
compromise the well-being of the patient. This includes, without
limitation, abnormal or uncontrollable cell growth, metastasis,
interference with the normal functioning of neighboring cells,
release of cytokines or other secretory products at abnormal
levels, suppression or aggravation of inflammatory or immunological
response, etc.
[0090] "Chronic" administration refers to administration of the
agent(s) in a continuous mode as opposed to an acute mode, so as to
maintain the initial therapeutic effect (activity) for an extended
period of time. "Intermittent" administration is treatment that is
not consecutively done without interruption, but rather is cyclic
in nature.
[0091] "Mammal" for purposes of treatment refers to any animal
classified as a mammal, including humans and other higher mammals,
domestic and farm animals, and zoo, sports, or pet animals, such as
dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc.
Preferably, the mammal is human.
[0092] Administration "in combination with" one or more further
therapeutic agents, or "co-administration," which terms are used
interchangeably, includes simultaneous (concurrent) and consecutive
administration in any order.
[0093] "Antibodies" (Abs) and "immunoglobulins" (Igs) are
glycoproteins having the same structural characteristics. While
antibodies exhibit binding specificity to a specific antigen,
immunoglobulins include both antibodies and other antibody-like
molecules which lack antigen specificity. Polypeptides of the
latter kind are, for example, produced at low levels by the lymph
system and at increased levels by myelomas.
[0094] "Native antibodies and immunoglobulins" are usually
heterotetrameric glycoproteins of about 150,000 daltons, composed
of two identical light (L) chains and two identical heavy (H)
chains. Each light chain is linked to a heavy chain by one covalent
disulfide bond, while the number of disulfide linkages varies
between the heavy chains of different immunoglobulin isotypes. Each
heavy and light chain also has regularly spaced intrachain
disulfide bridges. Each heavy chain has at one end a variable
domain (VH) followed by a number of constant domains. Each light
chain has a variable domain at one end (VL) and a constant domain
at its other end; the constant domain of the light chain is aligned
with the first constant domain of the heavy chain, and the light
chain variable domain is aligned with the variable domain of the
heavy chain. Particular amino acid residues are believed to form an
interface between the light- and heavy-chain variable domains
(Chothia et al., J. Mol. Biol. 186:651 [1985]; Novotny and Haber,
Proc. Natl. Acad. Sci. U.S.A. 82:4592 [1985]; Chothia et al.,
Nature 342: 877-883 [1989]).
[0095] The term "variable" refers to the fact that certain portions
of the variable domains differ extensively in sequence among
antibodies and are used in the binding and specificity of each
particular antibody for its particular antigen. However, the
variability is not evenly distributed throughout the variable
domains of antibodies. It is concentrated in three segments called
complementarity-determining regions (CDRs) or hypervariable regions
both in the light-chain and the heavy-chain variable domains. The
more highly conserved portions of variable domains are called the
framework (FR). The variable domains of native heavy and light
chains each comprise four FR regions, largely adopting a
.beta.-sheet configuration, connected by three CDRs, which form
loops connecting, and in some cases forming part of, the
.beta.-sheet structure. The CDRs in each chain are held together in
close proximity by the FR regions and, with the CDRs from the other
chain, contribute to the formation of the antigen-binding site of
antibodies (see Kabat et al. (1991) supra). The constant domains
are not involved directly in binding an antibody to an antigen, but
exhibit various effector functions, such as participation of the
antibody in antibody-dependent cellular toxicity.
[0096] The "light chains" of antibodies (immunoglobulins) from any
vertebrate species can be assigned to one of two clearly distinct
types, called .kappa. and .lambda., based on the amino acid
sequences of their constant domains.
[0097] Depending on the amino acid sequence of the constant domain
of their heavy chains, immunoglobulins can be assigned to different
classes. There are five major classes of immunoglobulins: IgA, IgD,
IgE, IgG, and IgM, and several of these can be further divided into
subclasses (isotypes), e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3,
IgG.sub.4, IgA.sub.1, and IgA.sub.2. The heavy-chain constant
domains that correspond to the different classes of immunoglobulins
are called .alpha., .delta., .epsilon. .gamma., and .mu.
respectively. The subunit structures and three-dimensional
configurations of different classes of immunoglobulins are well
known.
[0098] The term "antibody" includes all classes and subclasses of
intact immunoglobulins. The term "antibody" also covers antibody
fragments. The term "antibody" specifically covers monoclonal
antibodies, including antibody fragment clones.
[0099] "Antibody fragments" comprise a portion of an intact
antibody that contains the antigen binding or variable region of
the intact antibody. Examples of antibody fragments include Fab,
Fab', F(ab').sub.2, and Fv fragments; diabodies; single-chain
antibody molecules, including single-chain Fv (scFv) molecules; and
multispecific antibodies formed from antibody fragments.
[0100] The term "monoclonal antibody" as used herein refers to an
antibody (or antibody fragment) obtained from a population of
substantially homogeneous antibodies, i.e., the individual
antibodies comprising the population are identical except for
possible naturally occurring mutations that may be present in minor
amounts. Monoclonal antibodies are highly specific, being directed
against a single antigenic site. Furthermore, in contrast to
conventional (polyclonal) antibody preparations which typically
include different antibodies directed against different
determinants (epitopes), each monoclonal antibody is directed
against a single determinant on the antigen. In addition to their
specificity, the monoclonal antibodies are advantageous in that
they are synthesized by the hybridoma culture, and are not
contaminated by other immunoglobulins. The modifier "monoclonal"
indicates the character of the antibody as being obtained from a
substantially homogeneous population of antibodies, and is not to
be construed as requiring production of the antibody by any
particular method. For example, the monoclonal antibodies to be
used in accordance with the present invention may be made by the
hybridoma method first described by Kohler et al., Nature, 256:495
(1975), or may be made by recombinant DNA methods (see, e.g., U.S.
Pat. No. 4,816,567). The "monoclonal antibodies" also include
clones of antigen-recognition and binding-site containing antibody
fragments (Fv clones) isolated from phage antibody libraries using
the techniques described in Clackson et al., Nature, 352:624-628
(1991) and Marks et al., J. MoL Biol., 222:581-597 (1991), for
example.
[0101] The monoclonal antibodies herein specifically include
"chimeric" antibodies (immunoglobulins) in which a portion of the
heavy and/or light chain is identical with or homologous to
corresponding sequences in antibodies derived from a particular
species or belonging to a particular antibody class or subclass,
while the remainder of the chain(s) is identical with or homologous
to corresponding sequences in antibodies derived from another
species or belonging to another antibody class or subclass, as well
as fragments of such antibodies, so long as they exhibit the
desired biological activity (U.S. Pat. No. 4,816,567 to Cabilly et
al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855
[1984]).
[0102] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric immunoglobulins, immunoglobulin chains or fragments
thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
antigen-binding subsequences of antibodies) which contain minimal
sequence derived from non-human immunoglobulin. For the most part,
humanized antibodies are human immunoglobulins (recipient antibody)
in which residues from part or all of a complementarity-determining
region (CDR) of the recipient are replaced by residues from a CDR
of a non-human species (donor antibody) such as mouse, rat or
rabbit having the desired specificity, affinity, and capacity. In
some instances, Fv framework region (FR) residues of the human
immunoglobulin are replaced by corresponding non-human residues.
Furthermore, humanized antibodies may comprise residues which are
found neither in the recipient antibody nor in the imported CDR or
framework sequences. These modifications are made to further refine
and optimize antibody performance. In general, the humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin sequence. The humanized antibody
optimally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al., Nature,
321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988);
Presta, Curr. Op. Struct. Biol., 2:593-596 (1992); and Clark,
Immunol. Today 21: 397-402 (2000). The humanized antibody includes
a Primatized.TM. antibody wherein the antigen-binding region of the
antibody is derived from an antibody produced by immunizing macaque
monkeys with the antigen of interest.
[0103] "Single-chain Fv" or "scFv" antibody fragments comprise the
VH and VL domains of antibody, wherein these domains are present in
a single polypeptide chain. Generally, the scFv polypeptide further
comprises a polypeptide linker between the VH and VL domains, which
enables the scFv to form the desired structure for antigen binding.
For a review of scFv see Pluckthun, in The Pharmacology of
Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,
Springer-Verlag, New York, pp. 269-315 (1994), Dall'Acqua and
Carter, Curr. Opin. Struct. Biol. 8: 443-450 (1998), and Hudson,
Curr. Opin. Immunol. 11: 548-557 (1999).
[0104] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy-chain
variable domain (VH) connected to a light-chain variable domain
(VL) in the same polypeptide chain (VH-VL). By using a linker that
is too short to allow pairing between the two domains on the same
chain, the domains are forced to pair with the complementary
domains of another chain and create two antigen-binding sites.
Diabodies are described more fully in, for example, EP 404,097; WO
93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA,
90:6444-6448 (1993).
[0105] An "isolated" antibody is one which has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which would interfere with diagnostic or therapeutic uses
for the antibody, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred
embodiments, the antibody will be purified (1) to greater than 95%
by weight of antibody as determined by the Lowry method, and most
preferably more than 99% by weight, (2) to a degree sufficient to
obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue or, preferably, silver stain. Isolated antibody
includes the antibody in situ within recombinant cells since at
least one component of the antibody's natural environment will not
be present. Ordinarily, however, isolated antibody will be prepared
by at least one purification step.
[0106] By "neutralizing antibody" is meant an antibody molecule
which is able to eliminate or significantly reduce an effector
function of a target antigen to which it binds.
[0107] "Carriers" as used herein include pharmaceutically
acceptable carriers, excipients, or stabilizers which are nontoxic
to the cell or mammal being exposed thereto at the dosages and
concentrations employed. Often the physiologically acceptable
carrier is an aqueous pH buffered solution. Examples of
physiologically acceptable carriers include buffers such as
phosphate, citrate, and other organic acids; antioxidants including
ascorbic acid; low molecular weight (less than about 10 residues)
polypeptide; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, arginine or
lysine; monosaccharides, disaccharides, and other carbohydrates
including glucose, mannose, or dextrins; chelating agents such as
EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming
counterions such as sodium; and/or nonionic surfactants such as
TWEEN.TM., polyethylene glycol (PEG), and PLURONICS.TM..
[0108] The word "label" when used herein refers to a detectable
compound or composition which is conjugated directly or indirectly
to the antibody so as to generate a "labeled" antibody. The label
may be detectable by itself (e.g. radioisotope labels or
fluorescent labels) or, in the case of an enzymatic label, may
catalyze chemical alteration of a substrate compound or composition
which is detectable.
[0109] By "solid phase" is meant a non-aqueous matrix to which the
antibody of the present invention can adhere. Examples of solid
phases encompassed herein include those formed partially or
entirely of glass (e.g., controlled pore glass), polysaccharides
(e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol
and silicones. In certain embodiments, depending on the context,
the solid phase can comprise the well of an assay plate; in others
it is a purification column (e.g., an affinity chromatography
column). This term also includes a discontinuous solid phase of
discrete particles, such as those described in U.S. Pat. No.
4,275,149.
[0110] A "liposome" is a small vesicle composed of various types of
lipids, phospholipids and/or surfactant which is useful for
delivery of a drug (such as a PRO10282 polypeptide or antibody
thereto) to a mammal. The components of the liposome are commonly
arranged in a bilayer formation, similar to the lipid arrangement
of biological membranes.
[0111] A "small molecule" is defined herein to have a molecular
weight below about 500 Daltons.
[0112] The term "upregulation" is used in the broadest sense and
refers to the induction and/or enhancement of gene expression as
measured, for example, by quantification of mRNA levels.
[0113] The phrases "gene amplification" and "gene duplication" are
used interchangeably and refer to a process by which multiple
copies of a gene or gene fragment are formed in a particular cell
or cell line. The duplicated region (a stretch of amplified DNA) is
often referred to as "amplicon." Usually, the amount of the
messenger RNA (mRNA) produced, i.e., the level of gene expression,
also increases in the proportion of the number of copies made of
the particular gene expressed.
[0114] The term "anti-tumor agent" or "anti-cancer agent" is used
in the broadest sense and including any molecule useful in the
treatment of cancer. Such molecules include, without limitation,
polypeptides (including proteins), such as antibodies, peptides,
organic and inorganic small molecules, DNA and RNA molecules,
etc.
[0115] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. Without limitation, the term is
intended to include radioactive isotopes (e.g., I.sup.131,
I.sup.125, Y.sup.90, and Re.sup.186), chemotherapeutic agents, and
toxins such as enzymatically active toxins of bacterial, fungal,
plant or animal origin, or fragments thereof. The term specifically
includes maytansines and maytansinoids, BCNU, streptozoicin,
vincristine, and the family of agents described in U.S. Pat. Nos.
5,053,395 and 5,770,710, as well as esperamicins (U.S. Pat. No. 25
5,877,296).
[0116] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include adriamycin, doxorubicin, epirubicin, 5-fluorouracil,
cytosine arabinoside ("Ara-C"), cyclophosphamide, thiotepa,
busulfan, cytoxin, taxoids, e.g., paclitaxel (Taxol, Bristol-Myers
Squibb Oncology, Princeton, N.J.), and doxetaxel (Taxotere,
Rhne-Poulenc Rorer, Antony, Rnace), toxotere, methotrexate,
cisplatin, melphalan, vinblastine, bleomycin, etoposide,
ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine,
carboplatin, teniposide, daunomycin, carminomycin, aminopterin,
dactinomycin, mitomycins, esperamicins (see U.S. Pat. No.
4,675,187), 5-FU, 6-thioguanine, 6-mercaptopurine, actinomycin D,
VP-16, chlorambucil, melphalan, and other related nitrogen
mustards. Also included in this definition are hormonal agents that
act to regulate or inhibit hormone action on tumors such as
tamoxifen and onapristone.
[0117] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth of a cell, especially
cancer cell overexpressing any of the genes identified herein,
either in vitro or in vivo. Thus, the growth inhibitory agent is
one which significantly reduces the percentage of cells
overexpressing such genes in S phase. Examples of growth inhibitory
agents include agents that block cell cycle progression (at a place
other than S phase), such as agents that induce G1 arrest and
M-phase arrest. Classical M-phase blockers include the vincas
(vincristine and vinblastine), taxol, and topo II inhibitors such
as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.
Those agents that arrest G1 also spill over into S-phase arrest,
for example, DNA alkylating agents such as tamoxifen, prednisone,
dacarbazine, mechlorethamine, cisplatin, methotrexate,
5-fluorouracil, and ara-C. Further information can be found in The
Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1,
entitled "Cell cycle regulation, oncogens, and antineoplastic
drugs" by Murakami et al., (W B Saunders: Philadelphia, 1995),
especially p. 13.
[0118] "Doxorubicin" is an anthracycline antibiotic. The full
chemical name of doxorubicin is
(8S-cis)-10-[(3-amino-2,3,6-trideoxy-.alpha.-L-lyx-
o-hexapyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacety-
l)-1-methoxy-5,12-naphthacenedione.
[0119] The term "cytokine" is a generic term for proteins released
by one cell population which act on another cell as intercellular
mediators. Examples of such cytokines are lymphokines, monokines,
and traditional polypeptide hormones. Included among the cytokines
are growth hormone such as human growth hormone, N-methionyl human
growth hormone, and bovine growth hormone; parathyroid hormone;
thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein
hormones such as follicle stimulating hormone (FSH), thyroid
stimulating hormone (TSH), and luteinizing hormone (LH); hepatic
growth factor; fibroblast growth factor; prolactin; placental
lactogen; tumor necrosis factor-.alpha. and -.beta.;
mullerian-inhibiting substance; mouse gonadotropin-associated
peptide; inhibin; activin; vascular endothelial growth factor;
integrin; thrombopoietin (TPO); nerve growth factors such as NGF;
platelet-growth factor; transforming growth factors (TGFs) such as
TGF-.alpha. and TGF-.beta.; insulin-like growth factor-I and -II;
erythropoietin (EPO); osteoinductive factors; interferons such as
interferon-.alpha., -.beta., and -.gamma.; colony stimulating
factors (CSFs) such as macrophage-CSF (M-CSF);
granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);
interleukins (ILs) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor such
as TNF-.alpha. or TNF-.beta.; and other polypeptide factors
including LIF and kit ligand (KL). As used herein, the term
cytokine includes proteins from natural sources or from recombinant
cell culture and biologically active equivalents of the native
sequence cytokines.
[0120] The term "prodrug" as used in this application refers to a
precursor or derivative form of a pharmaceutically active substance
that is less cytotoxic to tumor cells compared to the parent drug
and is capable of being enzymatically activated or converted into
the more active parent form. See, e.g., Wilman, "Prodrugs in Cancer
Chemotherapy", Biochemical Society Transactions, 14:375-382, 615th
Meeting, Belfast (1986), and Stella et al., "Prodrugs: A Chemical
Approach to Targeted Drug Delivery", Directed Drug Delivery,
Borchardt et al., (ed.), pp. 147-267, Humana Press (1985). The
prodrugs of this invention include, but are not limited to,
phosphate-containing prodrugs, thiophosphate-containing prodrugs,
sulfate-containing prodrugs, peptide-containing prodrugs, D-amino
acid-modified Prodrugs, glysocylated prodrugs,
.beta.-lactam-containing prodrugs, optionally substituted
phenoxyacetamide-containing prodrugs or optionally substituted
phenylacetamide-containing prodrugs, 5-fluorocytosine and other
5-fluorouridine prodrugs which can be converted into the more
active cytotoxic free drug. Examples of cytotoxic drugs that can be
derivatized into a prodrug form for use in this invention include,
but are not limited to, those chemotherapeutic agents described
above.
[0121] An "effective amount" of a polypeptide disclosed herein or
an antagonist thereof, in reference to inhibition of neoplastic
cell growth, tumor growth or cancer cell growth, is an amount
capable of inhibiting, to some extent, the growth of target cells.
The term includes an amount capable of invoking a growth
inhibitory, cytostatic and/or cytotoxic effect and/or apoptosis of
the target cells. An "effective amount" of a polypeptide for the
purposes of inhibiting neoplastic cell growth, tumor cell or cancer
cell growth, may be determined empirically and in a routine
manner.
[0122] A "therapeutically effective amount", in reference to the
treatment of tumor, refers to an amount capable of invoking one or
more of the following effects: (1) inhibition, to some extent, of
tumor growth, including, slowing down and complete growth arrest;
(2) reduction in the number of tumor cells; (3) reduction in tumor
size; (4) inhibition (i.e., reduction, slowing down or complete
stopping) of tumor cell infiltration into peripheral organs; (5)
inhibition (i.e., reduction, slowing down or complete stopping) of
metastasis; (6) enhancement of anti-tumor immune response, which
may, but does not have to, result in the regression or rejection of
the tumor; and/or (7) relief, to some extent, of one or more
symptoms associated with the disorder. A "therapeutically effective
amount" of an anti-tumor agent may be determined empirically and in
a routine manner.
[0123] A "growth inhibitory amount" of an anti-tumor agent is an
amount capable of inhibiting the growth of a cell, especially
tumor, e.g. cancer cell, either in vitro or in vivo.
[0124] A "cytotoxic amount" of an anti-tumor agent is an amount
capable of causing the destruction of a cell, especially tumor,
e.g. cancer cell, either in vitro or in vivo. The "cytotoxic
amount" for purposes of inhibiting neoplastic cell growth may be
determined empirically and in a routine manner.
[0125] "Antisense oligodeoxynucleotides" or "antisense
oligonucleotides" (which terms are used interchangeably) are
defined as nucleic acid molecules that can inhibit the
transcription and/or translation of target genes in a
sequence-specific manner. The term "antisense" refers to the fact
that the nucleic acid is complementary t the coding ("sense")
genetic sequence of the target gene. Antisense oligonucleotides
hybridize in an antiparallel orientation to nascent mRNA through
Watson-Crick base-pairing. By binding the target mRNA template,
antisense oligonucleotides block the successful translation of the
encoded protein. The term specifically includes antisense agents
called "ribozomes" that have been designated to induce catalytic
cleavage of a target RNA by addition of a sequence that has natural
self-splicing activity (Warzocha and Wotowiec, "Antisense strategy"
biological utility and prospects int he treatment of hematological
malignancies." Leuk. Lymphoma 24:267-281 [1997]).
[0126] The term "therapeutic index" refers to the ratio between the
toxic concentration and the effective concentration of a drug, such
a therapeutic antibody.
[0127] B. Detailed Description
[0128] As discussed above, several components of the Wnt signaling
pathway have been implicated in human tumors or experimental cancer
models. Wnt-1 was found as an oncogene activated by the Mouse
Mammary Tumor Virus (MMTV) in murine breast cancer (Nusse and
Varmus, Cell 31:99-109 (1982)). Another member of the Wnt pathway,
the adenomatous polyposis coli (APC) tumor suppressor was first
isolated in human colon cancer (reviewed in Polakis, Biochim.
Biophys. Acta 1332(3):F127-47 (1997)). After establishing that APC
and .beta.-catenin bind each other, activating mutations in the
human .beta.-catenin gene were found in human colon cancer and
melanomas (Morin et al., Science 275:1752-53 (1997)). For review of
the role of the Wnt signaling in cancer see, e.g. Polakis, Genes
Dev. 14:1837-51 (2000) and Bienz and Clevers, Cell 103:311-20
(2000)).
[0129] The present invention concerns the enhancement of the
efficacy of treatment, such as immunotherapy, of tumors driven by
Wnt signaling.
[0130] 1. Identification of Gene Targets for Tumor Treatment
[0131] In one aspect of the present invention, drug targets for
tumor treatment are identified by analyzing differences in gene
expression resulting from retinoid treatment of tumor cells. In
particular, the invention concerns the identification of antigens
that are preferentially upregulated by the treatment of
Wnt-transformed cells with retinoids, such as retinoic acid, as
targets for cancer therapy. The preferred gene targets are those
which express cell surface proteins, and which are synergistically
upregulated by retinoid treatment and Wnt signaling.
[0132] a. Gene Expression Profiling
[0133] The most commonly used methods known in the art for the
quantification of mRNA expression in a sample include northern
blotting and in situ hybridization (Parker & Barnes, Methods in
Molecular Biology 106:247-283 (1999)); RNAse protection assays
(Hod, Biotechniques 13:852-854 (1992)); and reverse transcription
polymerase chain reaction (RT-PCR) (Weis et al., Trends in Genetics
8:263-264 (1992)). Alternatively, antibodies may be employed that
can recognize specific duplexes, including DNA duplexes, RNA
duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes.
[0134] Reverse Transcriptase PCR (RT-PCR)
[0135] Of the techniques listed above, the most sensitive and most
flexible quantitative method is RT-PCR, which can be used to
compare mRNA levels in different sample populations, in normal and
tumor tissues, with or without drug treatment, to characterize
patterns of gene expression, to discriminate between closely
related mRNAs, and to analyze RNA structure.
[0136] The first step is the isolation of mRNA from a target
sample. The starting material is typically total RNA isolated from
human tumors or tumor cell lines, and corresponding normal tissues
or cell lines, respectively. Thus RNA can be isolated from a
variety of primary tumors, including breast, lung, colon, prostate,
brain, liver, kidney, pancreas, spleen, thymus, testis, ovary,
uterus, etc., tumor, or tumor cell lines, with pooled DNA from
healthy donors. If the source of mRNA is a primary tumor, mRNA can
be extracted, for example, from frozen or archived
paraffin-embedded and fixed (e.g. formalin-fixed) tissue
samples.
[0137] Tumor cell lines, include, for example, human lung carcinoma
cell lines, such as A549 (SRCC768), Calu-1 (SRCC769), Calu-6
(SRCC770), H157 (SRCC771), H441 (SRCC772), H460 (SRCC773), SKMES-1
(SRCC774), SW900 (SRCC775), H522 (SRCC832), and H810 (SRCC833), all
available from ATCC. Primary human lung tumor cells usually derive
from adenocarcinomas, squamous cell carcinomas, large cell
carcinomas, non-small cell carcinomas, small cell carcinomas, and
broncho alveolar carcinomas, and include, for example, SRCC724
(adenocarcinoma, abbreviated as "AdenoCa") (LT1), SRCC725 (squamous
cell carcinoma, abbreviated as "SqCCa) (LT1a), SRCC726
(adenocarcinoma) (LT2), SRCC727 (adenocarcinoma) (LT3), SRCC728
(adenocarcinoma) (LT4), SRCC729 (squamous cell carcinoma) (LT6),
SRCC730 (adeno/squamous cell carcinoma) (LT7), SRCC731
(adenocarcinoma) (LT9), SRCC732 (squamous cell carcinoma) (LT10),
SRCC733 (squamous cell carcinoma) (LT11), SRCC734 (adenocarcinoma)
(LT12), SRCC735 (adeno/squamous cell carcinoma) (LT13), SRCC736
(squamous cell carcinoma) (LT15), SRCC737 (squamous cell carcinoma)
(LT16), SRCC738 (squamous cell carcinoma) (LT 17), SRCC739
(squamous cell carcinoma) (LT18), SRCC740 (squamous cell carcinoma)
(LT19), SRCC741 (lung cell carcinoma, abbreviated as "LCCa")
(LT21), SRCC811 (adenocarcinoma)(LT22), SRCC825(adenocarcinoma)
(LT8), SRCC886 (adenocarcinoma) (LT25), SRCC887 (squamous cell
carcinoma) (LT26), SRCC888 (adeno-BAC carcinoma) (LT27), SRCC889
(squamous cell carcinoma) (LT28), SRCC890 (squamous cell carcinoma)
(LT29), SRCC891 (adenocarcinoma) (LT30), SRCC892 (squamous cell
carcinoma) (LT31), SRCC894 (adenocarcinoma) (LT33). Also included
are human lung tumors designated SRCC1125 [HF-000631], SRCC1127
[HF-000641], SRCC1129 [HF-000643], SRCC1133 [HF-000840], SRCC1135
[HF-000842], SRCC1227 [HF-001291], SRCC1229 [HF-001293], SRCC1230
[HF-001294], SRCC1231 [HF-001295], SRCC1232 [HF-001296], SRCC1233
[HF-001297], SRCC1235 [HF-001299], and SRCC1236 [HF-001300].
[0138] Colon cancer cell lines include, for example, ATCC cell
lines SW480 (adenocarcinoma, SRCC776), SW620 (lymph node metastasis
of colon adenocarcinoma, SRCC777), Colo320 (carcinoma, SRCC778),
HT29 (adenocarcinoma, SRCC779), HM7 (a high mucin producing variant
of ATCC colon adenocarcinoma cell line, SRCC780, obtained from Dr.
Robert Warren, UCSF), CaWiDr (adenocarcinoma, SRCC781), HCT116
(carcinoma, SRCC782), SKCO1 (adenocarcinoma, SRCC783), SW403
(adenocarcinoma, SRCC784), LS174T (carcinoma, SRCC785), Colo205
(carcinoma, SRCC828), HCT15 (carcinoma, SRCC829), HCC2998
(carcinoma, SRCC830), and KM12 (carcinoma, SRCC831). Primary colon
tumors include colon adenocarcinomas designated CT2 (SRCC742), CT3
(SRCC743), CT8 (SRCC744), CT10 (SRCC745), CT12 (SRCC746), CT14
(SRCC747), CT15 (SRCC748), CT16 (SRCC749), CT17 (SRCC750), CT1
(SRCC751), CT4 (SRCC752), CT5 (SRCC753), CT6 (SRCC754), CT7
(SRCC755), CT9 (SRCC756), CT11 (SRCC757), CT18 (SRCC758), CT19
(adenocarcinoma, SRCC906), CT20 (adenocarcinoma, SRCC907), CT21
(adenocarcinoma, SRCC908), CT22 (adenocarcinoma, SRCC909), CT23
(adenocarcinoma, SRCC910), CT24 (adenocarcinoma, SRCC911), CT25
(adenocarcinoma, SRCC912), CT26 (adenocarcinoma, SRCC913), CT27
(adenocarcinoma, SRCC914),CT28 (adenocarcinoma, SRCC915), CT29
(adenocarcinoma, SRCC916), CT30 (adenocarcinoma, SRCC917), CT31
(adenocarcinoma, SRCC918), CT32(adenocarcinoma, SRCC919), CT33
(adenocarcinoma, SRCC920), CT35 (adenocarcinoma, SRCC921), and CT36
(adenocarcinoma, SRCC922). Also included are human colon tumors
designated SRCC1051 [HF-000499], SRCC1052 [HF-000539], SRCC1053
[HF-000575], SRCC1054 [HF-000698], SRCC1142 [HF-000762], SRCC1144
[HF-000789], SRCC1146 [HF-000795] and SRCC1148[HF-000811].
[0139] Human breast carcinoma cell lines include, for example,
HBL100 (SRCC759), MB435s (SRCC760), T47D (SRCC761), MB468
(SRCC762), MB175 (SRCC763), MB361 (SRCC764), BT20 (SRCC765), MCF7
(SRCC766), and SKBR3 (SRCC767), and human breast tumor center
designated SRCC1057 [HF-000545]. Also included are human breast
tumors designated SRCC1094, SRCC1095, SRCC1096, SRCC1097, SRCC1098,
SRCC1099, SRCC1100, SRCC1101, and human breast-met-lung-NS tumor
designated SRCC893 [LT 32].
[0140] Human kidney tumor centers include SRCC989 [HF-000611] and
SRCC1014 [HF-000613].
[0141] Human testis tumor center includes SRCC1001 [HF-000733] and
testis tumor margin SRCC999 [HF-000716].
[0142] Human parathyroid tumor includes SRCC1002 [HF-000831] and
SRCC1003 [HF-000832].
[0143] Methods for mRNA extraction are well known in the art and
are disclosed in standard textbooks of molecular biology, including
Ausubel et al., Current Protocols of Molecular Biology, John Wiley
and Sons (1997). Methods for RNA extraction from paraffin embedded
tissues are disclosed, for example, in Rupp and Locker, Lab Invest.
56:A67 (1987), and De Andrs et al., BioTechniques 18:42044 (1995).
In particular, RNA isolation can be performed using purification
kit, buffer set and protease from commercial manufacturers, such as
Qiagen, according to the manufacturer's instructions. For example,
total RNA from cells in culture can be isolated using Qiagen RNeasy
mini-columns. Total RNA from tissue samples can be isolated using
RNA Stat-60 (Tel-Test). RNA prepared from tumor can be isolated,
for example, by cesium chloride density gradient
centrifugation.
[0144] The present invention provides methods for identifying
genes, preferably cell surface antigens, the expression of which is
selectively enhanced by retinoid treatment in tumor cells driven by
Wnt signaling. Accordingly, a preferred source of the mRNA is a
tissue sample or cell line derived from a tumor the development
and/or progression of which is associated with defects in the Wnt
signaling pathway. As discussed above, for example, activating
mutations in .beta.-catenin have been identified in cancers of the
ovary and endometrium, Wilm's kidney tumors and melanomas,
demonstrating that defects in Wnt-1 signaling contribute to the
progression of these cancers (Kobayashi et al., Jpn J Cancer Res
90:55-9 (1999); Koesters et al., Cancer Res 59:3880-2 (1999);
Palacios and Hamallo, Cancer Res 58:1344-7 (1998); Rimm et al Am J
Pathol 154:325-9 (1999); Rubinfeld et al., Science 262:1731-1734
[1993]; Wright et al., Int J Cancer 82:625-9 (1999)). Similarly,
certain colorectal cancers are characterized by defective Wnt-1
signaling. Accordingly, frozen or paraffin-embedded samples or
tumor cell lines from such tumors are a preferred source of mRNA
for the expression profiling assays of the present invention.
Alternatively, tumor and normal cells engineered to conditionally
express a Wnt (e.g. Wnt-1) proto-oncogene can be a source of mRNA,
before and after retinoid treatment, and in the presence of absence
of Wnt (e.g. Wnt-1) expression.
[0145] As RNA cannot serve as a template for PCR, the first step in
gene expression profiling by RT-PCR is the reverse transcription of
the RNA template into cDNA, followed by its exponential
amplification in a PCR reaction. The two most commonly used reverse
transcriptases are avilo myeloblastosis virus reverse transcriptase
(AMV-RT) and Moloney murine leukemia virus reverse transcriptase
(MMLV-RT). The reverse transcription step is typically primed using
specific primers, random hexamers, or oligo-dT primers, depending
on the circumstances and the goal of expression profiling. For
example, extracted RNA can be reverse-transcribed using a GeneAmp
RNA PCR kit (Perkin Elmer, Calif., USA), following the
manufacturer's instructions. The derived cDNA can then be used as a
template in the subsequent PCR reaction.
[0146] Although the PCR step can use a variety of thermostable
DNA-dependent DNA polymerases, it typically employs the Taq DNA
polymerase, which has a 5'-3' nuclease activity but lacks a 3'-5'
proofreading endonuclease activity. Thus, TaqMan PCR typically
utilizes the 5'-nuclease activity of Taq or Tth polymerase to
hydrolyze a hybridization probe bound to its target amplicon, but
any enzyme with equivalent 5' nuclease activity can be used. Two
oligonucleotide primers are used to generate an amplicon typical of
a PCR reaction. A third oligonucleotide, or probe, is designed to
detect nucleotide sequence located between the two PCR primers. The
probe is non-extendible by Taq DNA polymerase enzyme, and is
labeled with a reporter fluorescent dye and a quencher fluorescent
dye. Any laser-induced emission from the reporter dye is quenched
by the quenching dye when the two dyes are located close together
as they are on the probe. During the amplification reaction, the
Taq DNA polymerase enzyme cleaves the probe in a template-dependent
manner. The resultant probe fragments disassociate in solution, and
signal from the released reporter dye is free from the quenching
effect of the second fluorophore. One molecule of reporter dye is
liberated for each new molecule synthesized, and detection of the
unquenched reporter dye provides the basis for quantitative
interpretation of the data.
[0147] TaqMan RT-PCR can be performed using commercially available
equipments, such as, for example, ABI PRIZM 7700.TM. Sequence
Detection System.TM. (Perkin-Elmer-Applied Biosystems, Foster City,
Calif., USA), or Lightcycler (Roche Molecular Biochemicals,
Mannheim, Germany). In a preferred embodiment, the 5' nuclease
procedure is run on a real-time quantitative PCR device such as the
ABI PRIZM 7700.TM. Sequence Detection System.TM.. The system
consists of a thermonuclear, laser, charge-coupled device (CCD),
camera and computer. The system amplifies samples in a 96-well
format on a thermocycler. During amplification, laser-induced
fluorescent signal is collected in real-time through fiber optics
cables for all 96 wells, and detected at the CCD. The system
includes software for running the instrument and for analyzing the
data.
[0148] 5'-Nuclease assay data are initially expressed as Ct, or the
threshold cycle. As discussed above, fluorescence values are
recorded during every cycle and represent the amount of product
amplified to that point in the amplification reaction. The point
when the fluorescent signal is first recorded as statistically
significant is the threshold cycle (C.sub.t). The .DELTA.Ct values
are used as quantitative measurement of the relative number of
starting copies of a particular target sequence in a nucleic acid
sample when comparing the expression of RNA in a cancer cell with
that from a normal cell.
[0149] To minimize errors and the effect of sample-to-sample
variation, RT-PCR is usually performed using an internal standard.
The ideal internal standard is expressed at a constant level among
different tissues, and is unaffected by the experimental treatment.
RNAs most frequently used to normalize patterns of gene expression
are mRNAs for the housekeeping genes
glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and
.beta.-actin.
[0150] Microarrays
[0151] Differential gene expression can also be identified, or
confirmed using the microarray technique. In this method,
nucleotide sequences of interest are plated, or arrayed, on a
microchip substrate. The arrayed sequences are then hybridized with
specific DNA probes from cells or tissues of interest. The genes
targeted by the present invention can be identified by constructing
normalized and subtracted cDNA libraries from mRNA extracted from
tumor cells or tissues, and the cells or tissue of healthy
subjects; purifying the DNA from the cDNA; treating both tumor and
healthy samples under otherwise identical conditions with a
retinoid; microarraying the purified DNA for expression analysis;
and probing microarrays to identify the genes from the clones that
are selectively upregulated by retinoid treatment in the tumor
cells or tissues, relative to corresponding normal cells or
tissues. Just as in the RT-PCR method, the source of mRNA typically
is total RNA isolated from human tumors or tumor cell lines, and
corresponding normal tissues or cell lines. Thus RNA can be
isolated from a variety of primary tumors or tumor cell lines,
including those listed above. If the source of mRNA is a primary
tumor, mRNA can be extracted, for example, from frozen or archived
paraffin-embedded and fixed (e.g. formalin-fixed) tissue samples,
which are routinely prepared and preserved in everyday clinical
practice.
[0152] In a specific embodiment of the microarray technique, PCR
amplified inserts of cDNA clones are applied to a substrate in a
dense array. Preferably at least 10,000 nucleotide sequences are
applied to the substrate. The microarrayed genes, immobilized on
the microchip at 10,000 elements each, are suitable for
hybridization under stringent conditions. Fluorescently labeled
cDNA probes may be generated through incorporation of fluorescent
nucleotides by reverse transcription of RNA extracted from tissues
of interest. Labeled cDNA probes applied to the chip hybridize with
specificity to each spot of DNA on the array. After stringent
washing to remove non-specifically bound probes, the chip is
scanned by confocal laser microscopy. Quantitation of hybridization
of each arrayed element allows for assessment of corresponding mRNA
abundance. With dual color fluorescence, separately labeled cDNA
probes generated from two sources of RNA are hybridized pairwise to
the array. The relative abundance of the transcripts from the two
sources corresponding to each specified gene is thus determined
simultaneously. The miniaturized scale of the hybridization affords
a convenient and rapid evaluation of the expression pattern for
large numbers of genes. Such methods have been shown to have the
sensitivity required to detect rare transcripts, which are
expressed at a few copies per cell, and to reproducibly detect at
least approximately two-fold differences in the expression levels
(Schena et al., Proc. Natl. Acad. Sci. USA 93(20):106-49
(1996)).
[0153] The methodology of hybridization of nucleic acids and
microarray technology is well known in the art. In one example, the
specific preparation of nucleic acids for hybridization and probes,
slides, and hybridization conditions are all detailed in PCT
Application Serial No. PCT/US01/10482, filed on Mar. 30, 2001, the
entire content of which is hereby expressly incorporated by
reference.
[0154] b. Retinoid Treatment
[0155] Regardless of the method chosen for gene expression
profiling, and to quantify mRNA transcription, the crust of the
present invention is the identification of genes the expression of
which is selectively enhanced by retinoid treatment in cancer cells
driven by Wnt signaling, relative to normal cells. In particular,
the gene expression profile of tumor cells characterized by the
involvement of the Wnt signaling pathway is determined in the
presence and absence of Wnt, e.g. Wnt-1 expression, and in the
presence and absence of retinoid treatment. After quantitation of
the gene expression data, genes are identified, the expression of
which is selectively upregulated by retinoid treatment of
Wnt-expressing tumor cells, relative to normal cells treated with
the same retinoid, and, preferably, also relative to tumor cells
not expressing Wnt, with or without retinoid treatment. As
discussed before, the retinoid may be a retinoic acid (also known
as tretinoin, vitamin A acid or vitamin A.sub.1), including both
all-trans-retinoic acid (all-trans-RA) and 9-cis-retinoic acid
(9-cis-RA), and retinoic acid derivatives consisting of four
isoprenoid units joined in a head-to-tail manner, such as retinol,
retinal, substituted retinoids, seco-, nor-, and retro-retinoids.
All retinoids may be formally derived from a monocyclic parent
compound containing five carbon-carbon double bonds and a
functional group at the terminus of the acyclic portion.
[0156] c. Cell-Based Tumor Assays
[0157] The gene targets identified in accordance with the present
invention and the treatment methods herein can be further validated
in cell-based tumor assays. The role of genes and gene products
identified herein in the development and pathology of tumor or
cancer can be tested by using primary tumor cells or cell lines
that have been identified to amplify the genes herein. Such cells
include, for example, the breast, colon and lung cancer cells and
cell lines listed above.
[0158] In a different approach, cells of a cell type known to be
involved in a particular tumor are transfected with the cDNAs
encoding the cell surface proteins identified herein, and the
ability of these cDNAs to induce excessive growth is analyzed.
Suitable cells include, for example, stable tumor cells lines such
as, the B104-1-1 cell line (stable NIH-3T3 cell line transfected
with the neu protooncogene) and ras-transfected NIH-3T3 cells,
which can be transfected with the desired gene, and monitored for
tumorigenic growth. Such transfected cell lines can then be used to
test the ability of anti-tumor agents, such as poly- or monoclonal
antibodies or antibody compositions in combination with retinoids
to inhibit tumorigenic cell growth by exerting cytostatic or
cytotoxic activity on the growth of the transformed cells, or by
mediating antibody-dependent cellular cytotoxicity (ADCC), in the
presence of absence of retinoid treatment. Cells transfected with
the coding sequences of the genes identified herein can further be
used to identify drug candidates for the treatment of cancer.
[0159] In addition, primary cultures derived from tumors in
transgenic animals (as described below) can be used in the
cell-based assays herein, although stable cell lines are preferred.
Techniques to derive continuous cell lines from transgenic animals
are well known in the art (see, e.g., Small et al., Mol. Cell.
Biol, 5:642-648 (1985)).
[0160] d. Animal Models
[0161] A variety of well known animal models can be used to further
understand the role of the genes identified herein in the
development and pathogenesis of tumors, and to test the efficacy of
retinoid treatment in combination with anticancer, e.g. antibody
therapy. The in vivo nature of such models makes them particularly
predictive of responses in human patients. Animal models of tumors
and cancers (e.g., breast cancer, colon cancer, prostate cancer,
lung cancer, etc.) include both non-recombinant and recombinant
(transgenic) animals. Non-recombinant animal models include, for
example, rodent, e.g., murine models. Such models can be generated
by introducing tumor cells into syngeneic mice using standard
techniques, e.g., subcutaneous injection, tail vein injection,
spleen implantation, intraperitoneal implantation, implantation
under the renal capsule, or orthopin implantation, e.g., colon
cancer cells implanted in colonic tissue. (See, e.g., PCT
publication No. WO 97/33551, published Sep. 18, 1997).
[0162] Probably the most often used animal species in oncological
studies are immunodeficient mice and, in particular, nude mice. The
observation that the nude mouse with hypoaplasia could successfully
act as a host for human tumor xenografts has lead to its widespread
use for this purpose. The autosomal recessive nu gene has been
introduced into a very large number of distinct congenic strains of
nude mouse, including, for example, ASW, A/He, AKR, BALB/c, B10.LP,
C17, C3H, C57BL, C57, CBA, DBA, DDD, I/st, NC, NFR, NFS, NFS/N,
NZB, NZC, NZW, P, RIII and SJL. In addition, a wide variety of
other animals with inherited immunological defects other than the
nude mouse have been bred and used as recipients of tumor
xenografts. For further details see, e.g., The Nude Mouse in
Oncology Research, E. Boven and B. Winograd, eds., CRC Press, Inc.,
1991.
[0163] The cells introduced into such animals can be derived from
known tumor/cancer cell lines, such as, for example, the Wnt-1
transfected murine C57MG breast epithelial cell line used in the
examples herein, stably expressing Wnt-1 (see also Diatchenko et
al., Proc. Natl. Acad. Sci. USA 93:6025-6030 (1996)), or from the
following ATCC human cell lines, after transfection with Wnt-1:
SW40, COLO320DM, HT-29, WiDr, and SW403 (colon adenocarcinomas),
SW620 (lympho node metastasis, colon adenocarcinoma), HT 116 (colon
carcinoma), SK-Co-1 (colon adenocarcinoma, ascites), and HM7 (a
variant of ATCC colon adenocarcinoma cell line LS 174T). Samples of
tumor or cancer cells can also be obtained from patients undergoing
surgery, using standard conditions, involving freezing and storing
in liquid nitrogen (Karmali et al., Br. J. Cancer, 48:689-696
(1983)), or from paraffin-embedded, formalin-fixed samples.
[0164] Tumor cells can be introduced into animals, such as nude
mice, by a variety of procedures. The subcutaneous (s.c.) space in
mice is very suitable for tumor implantation. Tumors can be
transplanted s.c. as solid blocks, as needle biopsies by use of a
trochar, or as cell suspensions. For solid block or trochar
implantation, tumor tissue fragments of suitable size are
introduced into the s.c. space. Cell suspensions are freshly
prepared from primary tumors or stable tumor cell lines, and
injected subcutaneously. Tumor cells can also be injected as
subdermal implants. In this location, the inoculum is deposited
between the lower part of the dermal connective tissue and the s.c.
tissue. Boven and Winograd (1991), supra.
[0165] Animal models of breast cancer can be generated, for
example, by implanting rat neuroblastoma cells (from which the neu
oncogene was initially isolated), or neu-transformed NIH-3T3 cells
into nude mice, essentially as described by Drebin et al., PNAS
USA, 83:9129-9133 (1986).
[0166] Similarly, animal models of colon cancer can be generated by
passaging colon cancer cells in animals, e.g., nude mice, leading
to the appearance of tumors in these animals. An orthotopic
transplant model of human colon cancer in nude mice has been
described, for example, by Wang et al., Cancer Research,
54:4726-4728 (1994) and Too et al., Cancer Research, 55:681-684
(1995). This model is based on the so-called "METAMOUSE" sold by
AntiCancer, Inc., (San Diego, Calif.).
[0167] Tumors that arise in animals can be removed and cultured in
vitro. Cells from the in vitro cultures can then be passaged to
animals. Such tumors can serve as targets for further testing or
drug screening. Alternatively, the tumors resulting from the
passage can be isolated and RNA from pre-passage cells and cells
isolated after one or more rounds of passage analyzed for
differential expression of genes of interest. Such passaging
techniques can be performed with any known tumor or cancer cell
lines.
[0168] For example, Meth A, CMS4, CMS5, CMS21, and WEHI-164 are
chemically induced fibrosarcomas of BALB/c female mice (DeLeo et
al., J. Exp. Med., 146:720 (1977)), which provide a highly
controllable model system for studying the anti-tumor activities of
various agents (Palladino et al., J. Immunol., 138:4023-4032
(1987)). Briefly, tumor cells are propagated in vitro in cell
culture. Prior to injection into the animals, the cell lines are
washed and suspended in buffer, at a cell density of about
10.times.10.sup.6 to 10.times.10.sup.7 cells/ml. The animals are
then infected subcutaneously with 10 to 100 .mu.l of the cell
suspension, allowing one to three weeks for a tumor to appear.
[0169] In addition, the Lewis lung (3LL) carcinoma of mice, which
is one of the most thoroughly studied experimental tumors, can be
used as an investigational tumor model. Efficacy in this tumor
model has been correlated with beneficial effects in the treatment
of human patients diagnosed with small cell carcinoma of the lung
(SCCL). This tumor can be introduced in normal mice upon injection
of tumor fragments from an affected mouse or of cells maintained in
culture (Zupi et al., Br. J. Cancer, 41:suppl. 4:309 (1980)), and
evidence indicates that tumors can be started from injection of
even a single cell and that a very high proportion of infected
tumor cells survive. For further information about this tumor model
see, Zacharski, Haemostasis, 16:300-320 (1986)).
[0170] One way of evaluating the efficacy of a test compound in an
animal model on an implanted tumor is to measure the size of the
tumor before and after treatment. Traditionally, the size of
implanted tumors has been measured with a slide caliper in two or
three dimensions. The measure limited to two dimensions does not
accurately reflect the size of the tumor, therefore, it is usually
converted into the corresponding volume by using a mathematical
formula. However, the measurement of tumor size is very inaccurate.
The therapeutic effects of a drug candidate can be better described
as treatment-induced growth delay and specific growth delay.
Another important variable in the description of tumor growth is
the tumor volume doubling time. Computer programs for the
calculation and description of tumor growth are also available,
such as the program reported by Rygaard and Spang-Thomsen, Proc.
6th Int. Workshop on Immune-Deficient Animals, Wu and Sheng eds.,
Basel, 1989, 301. It is noted, however, that necrosis and
inflammatory responses following treatment may actually result in
an increase in tumor size, at least initially. Therefore, these
changes need to be carefully monitored, by a combination of a
morphometric method and flow cytometric analysis.
[0171] Recombinant (transgenic) animal models can be engineered by
introducing the coding portion of the genes identified herein into
the genome of animals of interest, using standard techniques for
producing transgenic animals. Animals that can serve as a target
for transgenic manipulation include, without limitation, mice,
rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human
primates, e.g., baboons, chimpanzees and monkeys. Techniques known
in the art to introduce a transgene into such animals include
pronucleic microinjection (Hoppe and Wanger, U.S. Pat. No.
4,873,191); retrovirus-mediated gene transfer into germ lines
(e.g., Van der Putten et al., Proc. Natl. Acad. Sci. USA,
82:6148-615 (1985)); gene targeting in embryonic stem cells
(Thompson et al., Cell, 56:313-321 (1989)); electroporation of
embryos (Lo, Mol. Cell Biol., 3:1803-1814 (1983)); sperm-mediated
gene transfer (Lavitrano et al., Cell, 57:717-73 (1989)). For
review, see, for example, U.S. Pat. No. 4,736,866.
[0172] For the purpose of the present invention, transgenic animals
include those that carry the transgene only in part of their cells
("mosaic animals"). The transgene can be integrated either as a
single transgene, or in concatamers, e.g., head-to-head or
head-to-tail tandems. Selective introduction of a transgene into a
particular cell type is also possible by following, for example,
the technique of Lasko et al., Proc. Natl. Acad. Sci. USA,
89:6232-636 (1992).
[0173] The expression of the transgene in transgenic animals can be
monitored by standard techniques. For example, Southern blot
analysis or PCR amplification can be used to verify the integration
of the transgene. The level of mRNA expression can then be analyzed
using techniques such as in situ hybridization, Northern blot
analysis, PCR, or immunocytochemistry. The animals are further
examined for signs of tumor or cancer development.
[0174] Alternatively, "knock out" animals can be constructed which
have a defective or altered gene encoding a protein (e.g. a cell
surface protein) identified herein, as a result of homologous
recombination between the endogenous gene encoding the protein and
altered genomic DNA encoding the same protein introduced into an
embryonic cell of the animal. For example, cDNA encoding such
protein can be used to clone genomic DNA encoding the protein in
accordance with established techniques. A portion of the genomic
DNA encoding a particular protein can be deleted or replaced with
another gene, such as a gene encoding a selectable marker which can
be used to monitor integration. Typically, several kilobases of
unaltered flanking DNA (both at the 5' and 3' ends) are included in
the vector [see, e.g., Thomas and Capecchi, Cell, 51:503 (1987) for
a description of homologous recombination vectors]. The vector is
introduced into an embryonic stem cell line (e.g., by
electroporation) and cells in which the introduced DNA has
homologously recombined with the endogenous DNA are selected (see,
e.g., Li et al., Cell, 69:915 (1992)). The selected cells are then
injected into a blastocyst of an animal (e.g., a mouse or rat) to
form aggregation chimeras [see, e.g., Bradley, in Teratocarcinomas
and Embryonic Stem Cells: A Practical Approach, E. J. Robertson,
ed. (IRL, Oxford, 1987), pp. 113-152]. A chimeric embryo can then
be implanted into a suitable pseudopregnant female foster animal
and the embryo brought to term to create a "knock out" animal.
Progeny harboring the homologously recombined DNA in their germ
cells can be identified by standard techniques and used to breed
animals in which all cells of the animal contain the homologously
recombined DNA. Knockout animals can be characterized for instance,
by their ability to defend against certain pathological conditions
and by their development of pathological conditions due to absence
of the absent protein.
[0175] The efficacy of antibodies specifically binding the proteins
identified in accordance with the present invention and other drug
candidates, can be tested also in the treatment of spontaneous
animal tumors. A suitable target for such studies is the feline
oral squamous cell carcinoma (SCC). Feline oral SCC is a highly
invasive, malignant tumor that is the most common oral malignancy
of cats, accounting for over 60% of the oral tumors reported in
this species. It rarely metastasizes to distant sites, although
this low incidence of metastasis may merely be a reflection of the
short survival times for cats with this tumor. These tumors are
usually not amenable to surgery, primarily because of the anatomy
of the feline oral cavity. At present, there is no effective
treatment for this tumor. Prior to entry into the study, each cat
undergoes complete clinical examination, biopsy, and is scanned by
computed tomography (CT). Cats diagnosed with sublingual oral
squamous cell tumors are excluded from the study. The tongue can
become paralyzed as a result of such tumor, and even if the
treatment kills the tumor, the animals may not be able to feed
themselves. Each cat is treated repeatedly, over a longer period of
time. Photographs of the tumors will be taken daily during the
treatment period, and at each subsequent recheck. After treatment,
each cat undergoes another CT scan. CT scans and thoracic
radiograms are evaluated every 8 weeks thereafter. The data are
evaluated for differences in survival, response and toxicity as
compared to control groups. Positive response may require evidence
of tumor regression, preferably with improvement of quality of life
and/or increased life span.
[0176] In addition, other spontaneous animal tumors, such as
fibrosarcoma, adenocarcinoma, lymphoma, chrondroma, leiomyosarcoma
of dogs, cats, and baboons can also be tested. Of these mammary
adenocarcinoma in dogs and cats is a preferred model as its
appearance and behavior are very similar to those in humans.
However, the use of this model is limited by the rare occurrence of
this type of tumor in animals.
[0177] 2. Agents Targeting the Tumor Antigens Identified
[0178] The tumor antigens identified herein are desirable targets
for cancer therapy, since selective i;enhancement of their
expression by retinoid treatment relative to normal cells is
expected to improve the efficacy and therapeutic index of cancer
therapeutics directed against these antigens. This is generally
true for any antagonist of a tumor antigen identified in accordance
with the present invention.
[0179] a. Antagonists
[0180] Antagonists include oligonucleotides that bind to a tumor
antigen, and, in particular, antibodies including, without
limitation, poly- and monoclonal antibodies and antibody fragments,
single-chain antibodies, anti-idiotypic antibodies, and chimeric or
humanized versions of such antibodies or fragments, as well as
human antibodies and antibody fragments. Alternatively, a potential
antagonist may be a closely related protein, for example, a mutated
form of the tumor antigen that recognizes its receptor but imparts
no effect (e.g. a stra6, ephrin b1, 4-1BB ligand, autotaxin or ISRL
variant), thereby competitively inhibiting the action of the tumor
antigen.
[0181] Other antagonist may be an antisense RNA or DNA construct
prepared using antisense technology, where, e.g., an antisense RNA
or DNA molecule acts to block directly the translation of mRNA by
hybridizing to targeted mRNA and preventing protein translation.
Antisense technology can be used to control gene expression through
triple-helix formation or antisense DNA or RNA, both of which
methods are based on binding of a polynucleotide to DNA or RNA. For
example, the 5' coding portion of the polynucleotide sequence,
which encodes a mature tumor antigen herein, is used to design an
antisense RNA oligonucleotide of from about 10 to 40 base pairs in
length. A DNA oligonucleotide is designed to be complementary to a
region of the gene involved in transcription (triple helix-see, Lee
et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al., Science,
241: 456 (1988); Dervan et al., Science, 251:1360 (1991)), thereby
preventing transcription and the production of the target
polypeptide. The antisense RNA oligonucleotide hybridizes to the
mRNA in vivo and blocks translation of the mRNA molecule into the
target polypeptide (antisense-Okano, Neurochem., 56:560 (1991);
Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression
(CRC Press: Boca Raton, Fla., 1988). The oligonucleotides described
above can also be delivered to cells such that the antisense RNA or
DNA may be expressed in vivo to inhibit production of the target
polypeptide. When antisense DNA is used, oligodeoxyribonucleotides
derived from the translation-initiation site, e.g., between about
-10 and +10 positions of the target gene nucleotide sequence, are
preferred.
[0182] Antisense RNA or DNA molecules are generally at least about
5 bases in length, about 10 bases in length, about 15 bases in
length, about 20 bases in length, about 25 bases in length, about
30 bases in length, about 35 bases in length, about 40 bases in
length, about 45 bases in length, about 50 bases in length, about
55 bases in length, about 60 bases in length, about 65 bases in
length, about 70 bases in length, about 75 bases in length, about
80 bases in length, about 85 bases in length, about 90 bases in
length, about 95 bases in length, about 100 bases in length, or
more.
[0183] Potential antagonists include small molecules that bind to
the active site, the receptor binding site, or growth factor or
other relevant binding site of a tumor antigen identified herein,
thereby blocking its normal biological activity. Examples of small
molecules include, but are not limited to, small peptides or
peptide-like molecules, preferably soluble peptides, and synthetic
non-peptidyl organic or inorganic compounds. These small molecules
can be identified by screening techniques well known for those
skilled in the art.
[0184] Ribozymes are enzymatic RNA molecules capable of catalyzing
the specific cleavage of RNA. Ribozymes act by sequence-specific
hybridization to the complementary target RNA, followed by
endonucleolytic cleavage. Specific ribozyme cleavage sites within a
potential RNA target can be identified by known techniques. For
further details see, e.g., Rossi, Current Biology, 4:469-471
(1994), and PCT publication No. WO 97/33551 (published Sep. 18,
1997).
[0185] Nucleic acid molecules in triple-helix formation used to
inhibit transcription should be single-stranded and composed of
deoxynucleotides. The base composition of these oligonucleotides is
designed such that it promotes triple-helix formation via Hoogsteen
base-pairing rules, which generally require sizeable stretches of
purines or pyrimidines on one strand of a duplex. For further
details see, e.g., PCT publication No. WO 97/33551, supra.
[0186] In a preferred embodiment, the antagonists used in the
treatments methods of the present invention are antibodies that are
capable of specific binding a tumor antigen identified in
accordance with the present invention. Retinoid (e.g. retinoic
acid) treatment will selectively upregulate the tumor antigen
identified, and improve the efficacy and therapeutic index of
antibodies directed against such tumor antigens.
[0187] b. Antibodies
[0188] Methods of preparing polyclonal antibodies are known to the
skilled artisan. Polyclonal antibodies can be raised in a mammal,
for example, by one or more injections of an immunizing agent and,
if desired, an adjuvant. Typically, the immunizing agent and/or
adjuvant will be injected in the mammal by multiple subcutaneous or
intraperitoneal injections. The immunizing agent may include the
polypeptide identified in accordance with the present invention or
a fusion protein thereof. It may be useful to conjugate the
immunizing agent to a protein known to be immunogenic in the mammal
being immunized. Examples of such immunogenic proteins include but
are not limited to keyhole limpet hemocyanin, serum albumin, bovine
thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants
which may be employed include Freund's complete adjuvant and
MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose
dicorynomycolate). The immunization protocol may be selected by one
skilled in the art without undue experimentation.
[0189] Monoclonal Antibodies
[0190] The antibodies may, alternatively, be monoclonal antibodies.
Monoclonal antibodies may be prepared using hybridoma methods, such
as those described by Kohler and Milstein, Nature, 256:495 (1975).
In a hybridoma method, a mouse, hamster, or other appropriate host
animal, is typically immunized with an immunizing agent to elicit
lymphocytes that produce or are capable of producing antibodies
that will specifically bind to the immunizing agent. Alternatively,
the lymphocytes may be immunized in vitro.
[0191] The immunizing agent will typically include the polypeptide
to which the antibody binds or a fusion protein thereof. Generally,
either peripheral blood lymphocytes ("PBLs") are used if cells of
human origin are desired, or spleen cells or lymph node cells are
used if non-human mammalian sources are desired. The lymphocytes
are then fused with an immortalized cell line using a suitable
fusing agent, such as polyethylene glycol, to form a hybridoma cell
(Goding, Monoclonal Antibodies: Principles and Practice, Academic
Press, (1986) pp. 59-103). Immortalized cell lines are usually
transformed mammalian cells, particularly myeloma cells of rodent,
bovine and human origin. Usually, rat or mouse myeloma cell lines
are employed. The hybridoma cells may be cultured in a suitable
culture medium that preferably contains one or more substances that
inhibit the growth or survival of the unfused, immortalized cells.
For example, if the parental cells lack the enzyme hypoxanthine
guanine phosphoribosyl transferase (HGPRT or HPRT), the culture
medium for the hybridomas typically will include hypoxanthine,
aminopterin, and thymidine ("HAT medium"), which substances prevent
the growth of HGPRT-deficient cells.
[0192] Preferred immortalized cell lines are those that fuse
efficiently, support stable high level expression of antibody by
the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. More preferred immortalized cell lines
are murine myeloma lines, which can be obtained, for instance, from
the Salk Institute Cell Distribution Center, San Diego, Calif. and
the American Type Culture Collection, Manassas, Virginia. Human
myeloma and mouse-human heteromyeloma cell lines also have been
described for the production of human monoclonal antibodies
(Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal
Antibody Production Techniques and Applications, Marcel Dekker,
Inc., New York, (1987) pp. 51-63).
[0193] The culture medium in which the hybridoma cells are cultured
can then be assayed for the presence of monoclonal antibodies
directed against PRO10282. Preferably, the binding specificity of
monoclonal antibodies produced by the hybridoma cells is determined
by immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay
(ELISA). Such techniques and assays are known in the art. The
binding affinity of the monoclonal antibody can, for example, be
determined by the Scatchard analysis of Munson and Pollard, Anal.
Biochem., 107:220(1980).
[0194] After the desired hybridoma cells are identified, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods [Goding, supral]. Suitable culture media for this
purpose include, for example, Dulbecco's Modified Eagle's Medium
and RPMI-1640 medium. Alternatively, the hybridoma cells may be
grown in vivo as ascites in a mammal.
[0195] The monoclonal antibodies secreted by the subclones may be
isolated or purified from the culture medium or ascites fluid by
conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0196] The monoclonal antibodies may also be made by recombinant
DNA methods, such as those described in U.S. Pat. No. 4,816,567.
DNA encoding the monoclonal antibodies of the invention can be
readily isolated and sequenced using conventional procedures (e.g.,
by using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of murine
antibodies). The hybridoma cells of the invention serve as a
preferred source of such DNA. Once isolated, the DNA may be placed
into expression vectors, which are then transfected into host cells
such as simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of monoclonal antibodies in the recombinant
host cells. Prokaryotic hosts, such as E. coli are also suitable
for the recombinant production of the antibodies herein. The DNA
also may be modified, for example, by substituting the coding
sequence for human heavy and light chain constant domains in place
of the homologous murine sequences (U.S. Pat. No. 4,816,567;
Morrison et al., supra) or by covalently joining to the
immunoglobulin coding sequence all or part of the coding sequence
for a non-immunoglobulin polypeptide. Such a non-immunoglobulin
polypeptide can be substituted for the constant domains of an
antibody of the invention, or can be substituted for the variable
domains of one antigen-combining site of an antibody of the
invention to create a chimeric bivalent antibody.
[0197] The antibodies may be monovalent antibodies. Methods for
preparing monovalent antibodies are well known in the art. For
example, one method involves recombinant expression of
immunoglobulin light chain and modified heavy chain. The heavy
chain is truncated generally at any point in the Fc region so as to
prevent heavy chain crosslinking. Alternatively, the relevant
cysteine residues are substituted with another amino acid residue
or are deleted so as to prevent crosslinking.
[0198] In vitro methods are also suitable for preparing monovalent
antibodies. Digestion of antibodies to produce fragments thereof,
particularly, Fab fragments, can be accomplished using routine
techniques known in the art.
[0199] Human and Humanized Antibodies
[0200] The antibodies suitable for use in the treatment methods of
the invention may further comprise humanized antibodies or human
antibodies. Humanized forms of non-human (e.g., murine) antibodies
are chimeric immunoglobulin, immunoglobulin chains or fragments
thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
antigen-binding subsequences of antibodies) which contain minimal
sequence derived from non-human immunoglobulin. Humanized
antibodies include human immunoglobulins (recipient antibody) in
which residues from a complementary determining region (CDR) of the
recipient are replaced by residues from a CDR of a non-human
species (donor antibody) such as mouse, rat or rabbit having the
desired specificity, affinity and capacity. In some instances, Fv
framework residues of the human immunoglobulin are replaced by
corresponding non-human residues. Humanized antibodies may also
comprise residues which are found neither in the recipient antibody
nor in the imported CDR or framework sequences. In general, the
humanized antibody will comprise substantially all of at least one,
and typically two, variable domains, in which all or substantially
all of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin consensus sequence. The humanized
antibody optimally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin (Jones et al., Nature, 321:522-525 (1986); Riechmann
et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct.
Biol., 2:593-596 (1992)).
[0201] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source which is non-human.
These non-human amino acid residues are often referred to as
"import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers (Jones et al.,
Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327
(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by
substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized"
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567),
wherein substantially less than an intact human variable domain has
been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues
are substituted by residues from analogous sites in rodent
antibodies.
[0202] Human antibodies can also be produced using various
techniques known in the art, including phage display libraries
(Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et
al., J. Mol. Biol, 222:581 (1991)). The techniques of Cole et al.
and Boemer et al. are also available for the preparation of human
monoclonal antibodies (Cole et al., Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boemer et al., J.
Immunol., 147(1):86-95 (1991)). Similarly, human antibodies can be
made by introducing of human immunoglobulin loci into transgenic
animals, e.g., mice in which the endogenous immunoglobulin genes
have been partially or completely inactivated. Upon challenge,
human antibody production is observed, which closely resembles that
seen in humans in all respects, including gene rearrangement,
assembly, and antibody repertoire. This approach is described, for
example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;
5,625,126; 5,633,425; 5,661,016, and in the following scientific
publications: Marks et al., Bio/Technology 10:779-783 (1992);
Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature
368:812-13 (1994); Fishwild et al., Nature Biotechnology 14:845-51
(1996); Neuberger, Nature Biotechnology 14:826 (1996); Lonberg and
Huszar, Intern. Rev. Immunol. 13:65-93 (1995).
[0203] Bispecific Antibodies
[0204] Bispecific antibodies are monoclonal, preferably human or
humanized, antibodies that have binding specificities for at least
two different antigens. In the present case, one of the binding
specificities is for the target protein, the other one is for any
other antigen, and preferably for a cell-surface protein or
receptor or receptor subunit.
[0205] Methods for making bispecific antibodies are known in the
art. Traditionally, the recombinant production of bispecific
antibodies is based on the co-expression of two immunoglobulin
heavy-chain/light-chain pairs, where the two heavy chains have
different specificities (Milstein and Cuello, Nature, 305:537-539
(1983)). Because of the random assortment of immunoglobulin heavy
and light chains, these hybridomas (quadromas) produce a potential
mixture of ten different antibody molecules, of which only one has
the correct bispecific structure. The purification of the correct
molecule is usually accomplished by affinity chromatography steps.
Similar procedures are disclosed in WO 93/08829, published May 13,
1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).
[0206] Antibody variable domains with the desired binding
specificities (antibody-antigen combining sites) can be fused to
immunoglobulin constant domain sequences. The fusion preferably is
with an immunoglobulin heavy-chain constant domain, comprising at
least part of the hinge, CH2, and CH3 regions. It is preferred to
have the first heavy-chain constant region (CH1) containing the
site necessary for light-chain binding present in at least one of
the fusions. DNAs encoding the immunoglobulin heavy-chain fusions
and, if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are co-transfected into a suitable
host organism. For further details of generating bispecific
antibodies see, for example, Suresh et al., Methods in Enzymology,
121:210 (1986).
[0207] According to another approach described in WO 96/27011, the
interface between a pair of antibody molecules can be engineered to
maximize the percentage of heterodimers which are recovered from
recombinant cell culture. The preferred interface comprises at
least a part of the CH3 region of an antibody constant domain. In
this method, one or more small amino acid side chains from the
interface of the first antibody molecule are replaced with larger
side chains (e.g. tyrosine or tryptophan). Compensatory "cavities"
of identical or similar size to the large side chain(s) are created
on the interface of the second antibody molecule by replacing large
amino acid side chains with smaller ones (e.g. alanine or
threonine). This provides a mechanism for increasing the yield of
the heterodimer over other unwanted end-products such as
homodimers.
[0208] Bispecific antibodies can be prepared as full length
antibodies or antibody fragments (e.g. F(ab').sub.2 bispecific
antibodies). Techniques for generating bispecific antibodies from
antibody fragments have been described in the literature. For
example, bispecific antibodies can be prepared can be prepared
using chemical linkage. Brennan et al., Science 229:81 (1985)
describe a procedure wherein intact antibodies are proteolytically
cleaved to generate F(ab').sub.2 fragments. These fragments are
reduced in the presence of the dithiol complexing agent sodium
arsenite to stabilize vicinal dithiols and prevent intermolecular
disulfide formation. The Fab' fragments generated are then
converted to thionitrobenzoate (TNB) derivatives. One of the
Fab'-TNB derivatives is then reconverted to the Fab'-thiol by
reduction with mercaptoethylamnine and is mixed with an equimolar
amount of the other Fab'-TNB derivative to form the bispecific
antibody. The bispecific antibodies produced can be used as agents
for the selective immobilization of enzymes.
[0209] Fab' fragments may be directly recovered from E. coli and
chemically coupled to form bispecific antibodies. Shalaby et al.,
J. Exp. Med. 175:217-225 (1992) describe the production of a fully
humanized bispecific antibody F(ab').sub.2 molecule. Each Fab'
fragment was separately secreted from E. coli and subjected to
directed chemical coupling in vitro to form the bispecific
antibody. The bispecific antibody thus formed was able to bind to
cells overexpressing the ErbB2 receptor and normal human T cells,
as well as trigger the lytic activity of human cytotoxic
lymphocytes against human breast tumor targets.
[0210] Various technique for making and isolating bispecific
antibody fragments directly from recombinant cell culture have also
been described. For example, bispecific antibodies have been
produced using leucine zippers. Kostelny et al., J. Immunol.
148(5):1547-1553 (1992). The leucine zipper peptides from the Fos
and Jun proteins were linked to the Fab' portions of two different
antibodies by gene fusion. The antibody homodimers were reduced at
the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. This method can also be utilized for the
production of antibody homodimers. The "diabody" technology
described by Hollinger et al., Proc. Natl. Acad. Sci. USA
90:6444-6448 (1993) has provided an alternative mechanism for
making bispecific antibody fragments. The fragments comprise a
heavy-chain variable domain (V.sub.H) connected to a light-chain
variable domain (V.sub.L) by a linker which is too short to allow
pairing between the two domains on the same chain. Accordingly, the
V.sub.H and V.sub.L domains of one fragment are forced to pair with
the complementary V.sub.L and V.sub.H domains of another fragment,
thereby forming two antigen-binding sites. Another strategy for
making bispecific antibody fragments by the use of single-chain Fv
(sFv) dimers has also been reported. See, Gruber et al., J.
Immunol. 152:5368 (1994). Antibodies with more than two valencies
are contemplated. For example, trispecific antibodies can be
prepared. Tutt et al., J. Immunol. 147:60 (1991).
[0211] Exemplary bispecific antibodies may bind to two different
epitopes on a given polypeptide (e.g. cell surface antigen)
identified herein. Alternatively, one arm of the antibody, which
binds to target polypeptide, may be combined with an arm which
binds to a triggering molecule on a leukocyte such as a T-cell
receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for
IgG (Fc.gamma.R), such as Fc.gamma.RI (CD64), Fc.gamma.RII (CD32)
and Fc.gamma.RIII (CD16) so as to focus cellular defense mechanisms
to the cell expressing the particular polypeptide. Bispecific
antibodies may also be used to localize cytotoxic agents to cells
which express a particular antigen identified in accordance with
the present invention. These antibodies possess an antigen-binding
arm and an arm which binds a cytotoxic agent or a radionuclide
chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific
antibody of interest binds the target polypeptide and further binds
tissue factor (TF).
[0212] Heteroconjugate Antibodies
[0213] The use of heteroconjugate antibodies is also within the
scope of the present invention. Heteroconjugate antibodies are
composed of two covalently joined antibodies. Such antibodies have,
for example, been proposed to target immune system cells to
unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV
infection (WO 91/00360; WO 92/200373; EP 03089). It is contemplated
that the antibodies may be prepared in vitro using known methods in
synthetic protein chemistry, including those involving crosslinking
agents. For example, immunotoxins may be constructed using a
disulfide exchange reaction or by forming a thioether bond.
Examples of suitable reagents for this purpose include
iminothiolate and methyl-4-mercaptobutyrimidate and those
disclosed, for example, in U.S. Pat. No. 4,676,980.
[0214] Effector Function Engineering
[0215] It may be desirable to modify the antibody of the invention
with respect to effector function, so as to enhance, e.g., the
effectiveness of the antibody in treating cancer. For example,
cysteine residue(s) may be introduced into the Fc region, thereby
allowing interchain disulfide bond formation in this region. The
homodimeric antibody thus generated may have improved
internalization capability and/or increased complement-mediated
cell killing and antibody-dependent cellular cytotoxicity (ADCC).
See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J.
Immunol, 148: 2918-2922 (1992). Homodimeric antibodies with
enhanced anti-tumor activity may also be prepared using
heterobifunctional cross-linkers as described in Wolff et al Cancer
Research, 53: 2560-2565 (1993). Alternatively, an antibody can be
engineered that has dual Fc regions and may thereby have enhanced
complement lysis and ADCC capabilities. See Stevenson et al.,
Anti-Cancer Drug Design. 3: 219-230 (1989).
[0216] Immunoconjugates
[0217] The invention also includes the use of immunoconjugates
comprising an antibody conjugated to a cytotoxic agent such as a
chemotherapeutic agent, toxin (e.g., an enzymatically active toxin
of bacterial, fungal, plant, or animal origin, or fragments
thereof), or a radioactive isotope (i.e., a radioconjugate).
[0218] Chemotherapeutic agents useful in the generation of such
immunoconjugates have been described above. Enzymatically active
toxins and fragments thereof that can be used include diphtheria A
chain, nonbinding active fragments of diphtheria toxin, exotoxin A
chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin
proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),
momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes. A variety of
radionuclides are available for the production of radioconjugated
antibodies. Examples include .sup.212Bi, .sup.131I, .sup.131In,
.sup.90Y, and .sup.186Re.
[0219] Conjugates of the antibody and cytotoxic agent are made
using a variety of bifunctional protein-coupling agents such as
N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., Science, 238: 1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026.
[0220] In another embodiment, the antibody may be conjugated to a
"receptor" (such streptavidin) for utilization in tumor
pretargeting wherein the antibody-receptor conjugate is
administered to the patient, followed by removal of unbound
conjugate from the circulation using a clearing agent and then
administration of a "ligand" (e.g., avidin) that is conjugated to a
cytotoxic agent (e.g., a radionucleotide).
[0221] Preferred immunoconjugates are antibody-maytansinoid
conjugates. Immunoconjugates containing maytansinoids and their
therapeutic use are disclosed, for example, in U.S. Pat. Nos.
5,208,020; 5,416,064; and European Patent EP 0 425 235 B1, the
disclosures of which are hereby expressly incorporated by
reference. Liu et al., Proc Natl Acad Sci USA 93:8618-8623 (1996)
described immunoconjugates comprising a maytansinoid designated DM1
linked to the monoclonal antibody C242 directed against human
colorectal cancer. The conjugate was found to be highly cytotoxic
towards cultured colon cancer cells, and showed antitumor activity
in an in vivo tumor growth assay. Chari et al., Cancer Research
52:127-131 (1992) describe immunoconjugates in which a maytansinoid
was conjugated via a disulfide linked to the murine antibody A7
binding to an antigen on human colon cancer cell lines, or to
another murine monoclonal antibody TA. 1 that binds the HER-2/neu
oncogene.
[0222] The antibody-maytansinoid conjugates are prepared by
chemically linking an antibody to a maytansinoid molecule without
significantly diminishing the biological activity of either the
antibody or the maytansinoid molecule. An average of 3-4
maytansinoid molecules conjugates per antibody molecule has shown
efficacy in enhancing cytotoxicity of target cells without
negatively affecting the function or solubility of the antibody,
although even one molecule of toxin/antibody would be expected to
enhance cytotoxicity over the use of naked antibody. Maytansinoids
are well known in the art and can be synthesized by known
techniques or isolated from natural sources. Suitable maytansinoids
are disclosed, for example, in U.S. Pat. Nos 5,208,020; 4,137,230;
4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,747; 4,307,016;
4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821;
4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254;
4,362,663; and 4,371,533, the disclosures of which are hereby
expressly incorporated by reference. Preferred maytansinoids are
maytansinol and maytansinol analogues modified in the aromatic ring
or at other positions of the maytansinol molecule, such as various
maytansinol esters.
[0223] Another immunoconjugate of particular interest comprises an
antibody conjugated to one or more celicheamicin molecules. The
alicheamicin family of antibiotics are capable of producing
double-stranded DNA breaks at sub-picomolar concentrations. For the
preparation of conjugates of the calicheamicin family, see U.S.
Pat. Nos. 5,712,374; 5,714,586; 5,739,116; 5,767,285; 5,770,701;
5,773,001; and 5,877,296. Structural analogues of calicheamicin
which may be used include, but are not limited to,
.gamma..sub.1.sup.1,.alpha..sub.2.sup.1,-
.alpha..sub.3.sup.1,N-acetyl-.gamma..sub.1.sup.1, PSAG and
.theta..sup.1.sub.1 (Hinman et al., Cancer Res 53:3336-3342 (1993);
Lode et al., Cancer res 58:2925-2928 (1998) and the aforementioned
U.S. patents.
[0224] Another preferred anti-tumor drug that the antibody can be
conjugated to is QFA, an antifolate. Both calicheamicin and QFA
have intracellular sites of action and do not readily cross the
plasma membrane. Therefore, cellular uptake of these agents through
antibody mediated internalization greatly enhances their cytotoxic
effects.
[0225] The upregulation of the target polypeptides, e.g. cell
surface antigens, in accordance with the present invention lowers
the effective dose of the immunoconjugates and thus eliminates or
reduces the toxicity problems often associated with the
administration of cytotoxic agents or immunconjugates containing
cytotoxic agents.
[0226] Immunoliposomes
[0227] The antibodies used in the treatment methods of the present
invention may also be formulated as immunoliposomes. Liposomes
containing the antibody are prepared by methods known in the art,
such as described in Epstein et al., Proc. Natl. Acad. Sci. USA,
82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030
(1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with
enhanced circulation time are disclosed in U.S. Pat. No.
5,013,556.
[0228] Particularly useful liposomes can be generated by the
reverse-phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol, and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of the antibody of the present invention
can be conjugated to the liposomes as described in Martin et al .,
J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange
reaction. A chemotherapeutic agent (such as Doxorubicin) is
optionally contained within the liposome. See Gabizon et al., J.
National Cancer Inst., 81(19): 1484 (1989).
[0229] Antibody-Dependent Enzyme Mediated Prodrug Therapy
(ADEPT)
[0230] The antibodies herein may also be used in ADEPT by
conjugating the antibody to a prodrug-activating enzyme which
converts a prodrug (e.g., a peptidyl chemotherapeutic agent, see WO
81/01145) to an active anti-cancer drug. See, for example, WO
88/07378 and U.S. Pat. No. 4,975,278.
[0231] The enzyme component of the immunoconjugate useful for ADEPT
includes any enzyme capable of acting on a prodrug in such as way
so as to convert it into its more active, cytotoxic form.
[0232] Enzymes that are useful in the method of this invention
include, but are not limited to, glycosidase, glucose oxidase,
human lysosyme, human glucuronidase, alkaline phosphatase useful
for converting phosphate-containing prodrugs into free drugs;
arylsulfatase useful for converting sulfate-containing prodrugs
into free drugs; cytosine deaminase useful for converting non-toxic
5-fluorocytosine into the anti-cancer drug 5-fluorouracil;
proteases, such as serratia protease, thermolysin, subtilisin,
carboxypeptidases (e.g., carboxypeptidase G2 and carboxypeptidase
A) and cathepsins (such as cathepsins B and L), that are useful for
converting peptide-containing prodrugs into free drugs;
D-alanylcarboxypeptidases, useful for converting prodrugs that
contain D-amino acid substituents; carbohydrate-cleaving enzymes
such as .beta.-galactosidase and neuraminidase useful for
converting glycosylated prodrugs into free drugs; .beta.-lactamase
useful for converting drugs derivatized with .beta.-lactams into
free drugs; and penicillin amidases, such as penicillin Vamidase or
penicillin G amidase, useful for converting drugs derivatized at
their amine nitrogens with phenoxyacetyl or phenylacetyl groups,
respectively, into free drugs. Alternatively, antibodies with
enzymatic activity, also known in the art as "abzymes" can be used
to convert the prodrugs of the invention into free active drugs
(see, e.g., Massey, Nature, 328:457-458 (1987)). Antibody-abzyme
conjugates can be prepared as described herein for delivery of the
abzyme to a tumor cell population.
[0233] The enzymes can be covalently bound to the antibodies by
techniques well known in the art such as the use of the
heterobifunctional cross-linking agents discussed above.
Alternatively, fusion proteins comprising at least the antigen
binding region of the antibody of the invention linked to at least
a functionally active portion of an enzyme of the invention can be
constructed using recombinant DNA techniques well known in the art
(see, e.g., Neuberger et al., Nature, 312:604-608 (1984)).
[0234] 3. Pharmaceutical Compositions
[0235] The antibodies specifically binding the tumor antigens
identified in accordance with the present invention, as well as
other molecules targeting such tumor antigens, can be administered
for the treatment of various disorders in the form of
pharmaceutical compositions.
[0236] If the target polypeptide (tumor antigen) is intracellular
and whole antibodies are used as inhibitors, internalizing
antibodies are preferred. However, lipofections or liposomes can
also be used to deliver the antibody, or an antibody fragment, into
cells. Where antibody fragments are used, the smallest inhibitory
fragment that specifically binds to the binding domain of the
target protein is preferred. For example, based upon the
variable-region sequences of an antibody, peptide molecules can be
designed that retain the ability to bind the target protein
sequence. Such peptides can be synthesized chemically and/or
produced by recombinant DNA technology. See, e.g., Marasco et al.,
Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993). The formulation
herein may also contain more than one active compound as necessary
for the particular indication being treated, preferably those with
complementary activities that do not adversely affect each other.
Alternatively, or in addition, the composition may comprise an
agent that enhances its function, such as, for example, a cytotoxic
agent, cytokine, chemotherapeutic agent, or growth-inhibitory
agent. Such molecules are suitably present in combination in
amounts that are effective for the purpose intended.
[0237] The active ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles, and nanocapsules) or in macroemulsions. Such
techniques are disclosed in Remington's Pharmaceutical Sciences,
supra.
[0238] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0239] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g., films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods. When encapsulated antibodies remain in
the body for a long time, they may denature or aggregate as a
result of exposure to moisture at 37.degree. C., resulting in a
loss of biological activity and possible changes in immunogenicity.
Rational strategies can be devised for stabilization depending on
the mechanism involved. For example, if the aggregation mechanism
is discovered to be intermolecular S--S bond formation through
thio-disulfide interchange, stabilization may be achieved by
modifying sulfhydryl residues, lyophilizing from acidic solutions,
controlling moisture content, using appropriate additives, and
developing specific polymer matrix compositions.
[0240] 4. Treatment Methods
[0241] It is contemplated that the antibodies and other anti-tumor
compounds of the present invention may be used to treat various
conditions, including those characterized by overexpression and/or
activation of the tumor antigens identified herein. Exemplary
conditions or disorders to be treated with such antibodies and
other compounds, including, but not limited to, small organic and
inorganic molecules, peptides, antisense molecules, etc., include
benign or malignant tumors (e.g., renal, liver, kidney, bladder,
breast, gastric, ovarian, colorectal, prostate, pancreatic, lung,
vulval, thyroid, hepatic carcinomas; sarcomas; glioblastomas; and
various head and neck tumors); leukemias and lymphoid malignancies;
other disorders such as neuronal, glial, astrocytal, hypothalamic
and other glandular, macrophagal, epithelial, stromal and
blastocoelic disorders; and inflammatory, angiogenic and
immunologic disorders. Particularly preferred targets for treatment
with antibodies and other antagonists of the present invention are
tumors that harbor genetic defects in the Wnt-1 pathway (e.g.
leading to abnormal activation of .beta.-catenin signaling) and/or
overexpress a tumor antigen identified herein. Thus, for example,
it has been found that human cancers harboring genetic defects in
the Wnt-1 pathway typically also exhibit overexpression of the
tumor antigen Stra6, but not all Stra6 overexpressing tumors have
been known to be associated with mutations in the Wnt-1 pathway.
The antagonists of the present invention have great potential in
the treatment of tumors characterized by aberrant Wnt signaling,
especially when the target antigen is characterized by synergistic
enhancement of its expression by a combination of Wnt-1 and
retinoid treatment. A preferred group of such tumors includes
colorectal tumors, ovary, endometrium, and Wilm's kidney tumor,
breast cancer, prostate cancer, gastric cancer, lung cancer,
hepatocellular cancer, melanoma, and pheochromocytome (a tumor
derived from the adrenal medulla).
[0242] The anti-tumor agents of the present invention, e.g.,
antibodies, are administered to a mammal, preferably a human, in
combination with a retinoid, e.g., retinoic acid, in accord with
known methods, such as intravenous administration as a bolus or by
continuous infusion over a period of time, by intramuscular,
intraperitoneal, intracerobrospinal, subcutaneous, intra-articular,
intrasynovial, intrathecal, oral, topical, or inhalation routes.
Intravenous administration of the antibody is preferred. Retinoids
can be administered by the same of different routes of
administration. In Example 15 all-trans-retinoi aid (ATRA) has been
shown to be effective when administered orally. Based on this
experimental finding, and also in view of its convenience, oral
administration of the retinoids is preferred. Alternatively or in
addition, the retinoids may also be administered by peri-tumoral
injection.
[0243] Retinoid treatment may precede or follow the treatment with
the anti-tumor agent, e.g., antibody, or may occur simultaneously.
Since the primary goal of the retinoid treatment is to upregulate
the target antigen in order to enhance the efficacy of tumor
therapy, the retinoid is preferably administered prior to or
simultaneously with treatment with an anti-tumor agent. In the case
of concurrent treatment, the anti-tumor agent and the retinoid may
be formulated separately, or may be included in the same
pharmaceutical composition. The effective amount of a retinoid can
be determined by routine testing, and typically is between about 15
mg/kg and about 400 mg/kg, preferably between about 20 kg/kg and
about 200 mg/kg, more preferably between about 25 mg/kg and about
150 mg/kg of body weight. In a particularly preferred embodiment,
the dosage range of the retinoid, e.g. ATRA, is about 15-75
mg/m.sup.2/day. In addition, the treatment may involve the
administration of a Wnt, e.g. Wnt-1, since the coadministration of
a retinoid and Wnt, e.g. Wnt-1 may synergistically induce the genes
identified herein. Coadministration includes simultaneous
administration, or consecutive administration of the two agents in
either order.
[0244] Currently, depending on the stage of the cancer, cancer
treatment involves one or a combination of the following therapies:
surgery to remove the cancerous tissue, radiation therapy, and
chemotherapy. The treatment methods of the present invention
improve the therapeutic index of anti-tumor agents, such as
antibodies, and thereby allow the reduction of the effective dose
to be administered. Accordingly, the therapeutic methods herein are
especially useful in the treatment of elderly patients and others
who do not tolerate well the toxicity and side effects of
chemotherapy and in metastatic disease where radiation therapy has
limited usefulness.
[0245] Other therapeutic regimens may be combined with the
administration of the anti-cancer agents, e.g., antibodies and
retinoids. For example, the patient to be treated with such
anti-cancer agents may also receive radiation therapy and/or may
undergo surgery. Alternatively, or in addition, a chemotherapeutic
agent may be administered to the patient. Preparation and dosing
schedules for such chemotherapeutic agents may be used according to
manufacturers'instructions or as determined empirically by the
skilled practitioner. Preparation and dosing schedules for such
chemotherapy are also described in Chemotherapy Service Ed., M. C.
Perry, Williams & Wilkins, Baltimore, Md. (1992). The
chemotherapeutic agent may precede, or follow administration of the
anti-tumor agent, e.g., antibody, or may be given simultaneously
therewith. The antibody may be combined with an anti-estrogen
compound such as tamoxifen or an anti-progesterone such as
onapristone (see, EP 616812) in dosages known for such
molecules.
[0246] It may be desirable to also administer antibodies against
other tumor associated antigens, such as antibodies which bind to
the ErbB2, EGFR, ErbB3, ErbB4, or vascular endothelial factor
(VEGF). Alternatively, or in addition, two or more antibodies
binding the same or two or more different antigens disclosed herein
may be co-administered to the patient. Sometimes, it may be
beneficial to also administer one or more cytokines to the patient.
In a preferred embodiment, the antibodies herein are
co-administered with a growth inhibitory agent. For example, the
growth inhibitory agent may be administered first, followed by an
antibody of the present invention. However, simultaneous
administration or administration of the antibody of the present
invention first is also contemplated. Suitable dosages for the
growth inhibitory agent are those presently used and may be lowered
due to the combined action (synergy) of the growth inhibitory agent
and the antibody herein.
[0247] For the prevention or treatment of disease, the appropriate
dosage of an anti-tumor agent, e.g., an antibody herein will depend
on the type of disease to be treated, as defined above, the
severity and course of the disease, whether the agent is
administered for preventive or therapeutic purposes, previous
therapy, the patient's clinical history and response to the agent,
and the discretion of the attending physician. The agent is
suitably administered to the patient at one time or over a series
of treatments.
[0248] For example, depending on the type and severity of the
disease, about 1 .mu.g/kg to 15 mg/kg (e.g., 0.1-20 mg/kg) of
antibody is an initial candidate dosage for administration to the
patient, whether, for example, by one or more separate
administrations, or by continuous infusion. A typical daily dosage
might range from about 1 .mu.g/kg to 100 mg/kg or more, depending
on the factors mentioned above. For repeated administrations over
several days or longer, depending on the condition, the treatment
is sustained until a desired suppression of disease symptoms
occurs. However, other dosage regimens may be useful. The progress
of this therapy is easily monitored by conventional techniques and
assays.
[0249] 5. Articles of Manufacture
[0250] In another embodiment of the invention, an article of
manufacture containing the antibodies or other antagonist herein,
in combination with a retinoid, are provided. The article of
manufacture comprises a container and a label. Suitable containers
include, for example, bottles, vials, syringes, and test tubes. The
containers may be formed from a variety of materials such as glass
or plastic. The container holds a composition which is effective
for treating a condition targeted and may have a sterile access
port (for example the container may be an intravenous solution bag
or a vial having a stopper pierceable by a hypodermic injection
needle). The active agent in the composition is usually an
anti-tumor agent capable of interfering with the activity of a gene
product identified herein, e.g., an antibody. The label on, or
associated with, the container indicates that the composition is
used for diagnosing or treating the condition of choice. The
article of manufacture will further comprise, within the same or a
separate container, a retinoid and optionally a
pharmaceutically-acceptable buffer, such as phosphate-buffered
saline, Ringer's solution and dextrose solution. It may further
include other materials desirable from a commercial and user
standpoint, including other buffers, diluents, filters, needles,
syringes, and package inserts with instructions for use.
[0251] 6. Diagnostic Methods
[0252] While cell surface proteins, such as growth receptors
overexpressed in certain tumors are excellent targets for drug
candidates or tumor (e.g., cancer) treatment, the same proteins
along with secreted proteins encoded by the genes amplified in
tumor cells find additional use in the diagnosis and prognosis of
tumors. For example, antibodies directed against the protein
products of genes amplified in tumor cells can be used as tumor
diagnostics or prognostics.
[0253] For example, antibodies, including antibody fragments, can
be used to qualitatively or quantitatively detect the expression of
proteins encoded by the amplified genes ("marker gene products").
The antibody preferably is equipped with a detectable, e.g.,
fluorescent label, and binding can be monitored by light
microscopy, flow cytometry, fluorimetry, or other techniques known
in the art. These techniques are particularly suitable, if the
amplified gene encodes a cell surface protein, e.g., a growth
factor. Such binding assays are performed essentially as described
hereinabove.
[0254] The diagnostic assays of the present invention take
advantage of the finding that retinoids, e.g. retinoic acid,
selectively upregulate the tumor antigens identified herein.
Accordingly, the diagnostic assays are performed in conjunction
with retinoid treatment, where the retinoid treatment may precede,
occur concurrently with, or follow the administration of the
diagnostic agent targeting a tumor antigen, such as a corresponding
antibody. This selective upregulation of the tumor antigen by
retinoid treatment greatly improves the sensitivity of the
diagnostic assay, and may allow the detection of tumor antigens
which would otherwise not be detectable. This, in turn, allows
early detection of the tumor, and early intervention using the most
appropriate treatment, including targeting the tumor antigen
identified.
[0255] In situ detection of antibody binding to the marker gene
products can be performed, for example, by immunofluorescence or
immunoelectron microscopy. For this purpose, a histological
specimen is removed from the patient, and a labeled antibody is
applied to it, preferably by overlaying the antibody on a
biological sample. This procedure also allows for determining the
distribution of the marker gene product in the tissue examined. It
will be apparent for those skilled in the art that a wide variety
of histological methods are readily available for in situ
detection.
[0256] The following examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present
invention in any way.
[0257] All patent and literature references cited in the present
specification are hereby incorporated by reference in their
entirety.
EXAMPLES
[0258] Commercially available reagents referred to in the examples
were used according to manufacturer's instructions unless otherwise
indicated. The source of those cells identified in the following
examples, and throughout the specification, by ATCC accession
numbers is the American Type Culture Collection, Manassas, Va. In
the following Examples, unless otherwise specified, "Sra6" will
refer to native sequence PRO10282 polypeptide.
Example 1
Isolation of cDNA Clones Encoding a Human PRO10282 and PRO19578
polypeptides
[0259] cDNA clones (DNA148380-2827 and DNA148389-2827-1) encoding
native human PRO10282 and PRO19578 polypeptides were identified
using a yeast screen, in a human fetal brain cDNA library that
preferentially represents the 5' ends of the primary cDNA
clones.
[0260] Clone DNA148380-2827 contains a single open reading frame
with an apparent translational initiation site at nucleotide
positions 49-51 and ending at the stop codon at nucleotide
positions 2050-2052 (FIG. 1). The predicted polypeptide precursor
is 667 amino acids long (FIG. 2). The full-length PRO10282 protein
shown in FIG. 2 has an estimated molecular weight of about 73502
daltons and a pI of about 9.26. Analysis of the full-length
PRO10282 sequence shown in FIG. 2 (SEQ ID NO: 2) evidences the
presence of a variety of important polypeptide domains as shown in
FIG. 2, wherein the locations given for those important polypeptide
domains are approximate as described above. Clone DNA148380-2827
has been deposited with ATCC on Jan. 11, 2000 and is assigned ATCC
Deposit No. PTA-1181.
[0261] Clone DNA148389-2827-1 contains a single open reading frame
with an apparent translational initiation site at nucleotide
positions 186-188 and ending at the stop codon at nucleotide
positions 2160-2162 (FIG. 6, SEQ ID NO: 4). The predicted
polypeptide precursor is 658 amino acids long (FIG. 7, SEQ ID NO:
5). The full-length PRO19578 protein shown in FIG. 7 has an
estimated molecular weight of about 72583 daltons and a pI of about
9.36. Analysis of the full-length PRO19578 sequence shown in FIG. 7
(SEQ ID NO: 5) evidences the presence of a variety of important
polypeptide domains as shown in FIG. 7, wherein the locations given
for those important polypeptide domains are approximate as
described above. Noteworthy is the presence of nine potential
transmembrane domains and fourteen cysteine residues conserved
between the human and the corresponding mouse sequence. While mouse
Stra6 has three potential N-linked glycosylation sites, the human
PRO19578 (native human Stra6) polypeptide has one. Clone
148389-2827-1 has been deposited with ATCC on Feb. 23, 2000, and is
assigned ATCC Deposit No. PTA-1402.
[0262] An analysis of the Dayhoff database (version 35.45 SwissProt
35), using the ALIGN-2 sequence alignment analysis of the
full-length sequence shown in FIG. 2 (SEQ ID NO: 2), and of the
full-length sequence shown in FIG. 7 (SEQ ID NO: 5) evidenced
sequence identity between the PRO10282 amino acid sequence and the
following Dayhoff sequences: AF062476, P_W88559 and HGS_RE259.
[0263] As shown in FIG. 8, comparison of the full-length human
PRO10282 and PRO19578 polypeptides shows that PRO19578 contains a
deletion of nine amino acids (SPVDFLAGD; SEQ ID NO: 13) at
positions 89-97 of the PRO10282 amino acid sequence. In addition,
PRO19578 contains an isoleucine (I) at amino acid position 518 in
place of methionine (M) at the corresponding position (position
527) of PRO10282, which results from a G/A polymorphism at this
position. Both the PRO10282 and the native sequence PRO19578
polypeptides are believed to be the human homologues of the mouse
Stra6 polypeptide, and are, therefore, also referred to as "Stra6."
Mouse Stra6 and the native sequence human full-length PRO10282
polypeptide encoded by DNA148340-2827 show about 74% amino acid
sequence identity.
[0264] FIG. 9 shows the hydrophobicity plot of the native sequence
human full-length PRO10282 polypeptide encoded by DNA148380-2827,
briefly referred to as "human Stra6." As shown in FIG. 9, about 50%
of the amino acid residues in this 667 amino acids long polypeptide
are hydrophobic.
[0265] The human Stra6 gene was localized to chromosome 15q23 as
determined by UNIGENE. Preliminary fine mapping indicates that
Stra6 is located in the STS interval D15S124-D15S160 and the
GeneMap'98 position corresponds to 244.52 on the G3 map.
Example 2
Use of PRO10282 and PRO19578 as a Hybridization Probe
[0266] The following method describes use of a nucleotide sequence
encoding PRO10282 and PRO19578 as a hybridization probe.
[0267] DNA comprising the coding sequence of full-length or mature
PRO10282 or PRO19578 is employed as a probe to screen for
homologous DNAs (such as those encoding naturally-occurring
variants of PRO10282 or PRO19578) in human tissue cDNA libraries or
human tissue genomic libraries.
[0268] Hybridization and washing of filters containing either
library DNAs is performed under the following high stringency
conditions. Hybridization of radiolabeled PRO10282-derived probe to
the filters is performed in a solution of 50% formamide,
5.times.SSC, 0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium
phosphate, pH 6.8, 2.times.Denhardt's solution, and 10% dextran
sulfate at 42.degree. C. for 20 hours. Washing of the filters is
performed in an aqueous solution of 0.1.times.SSC and 0.1% SDS at
42.degree. C.
[0269] DNAs having a desired sequence identity with the DNA
encoding full-length native sequence PRO10282 or PRO19578 can then
be identified using standard techniques known in the art.
Example 3
Expression of PRO10282 and PRO19578 in E. coli
[0270] This example illustrates preparation of an unglycosylated
form of PRO10282 or PRO19578 by recombinant expression in E.
coli.
[0271] The DNA sequence encoding PRO10282 or PRO19578 is initially
amplified using selected PCR primers. The primers should contain
restriction enzyme sites which correspond to the restriction enzyme
sites on the selected expression vector. A variety of expression
vectors may be employed. An example of a suitable vector is pBR322
(derived from E. coli; see Bolivar et al., Gene, 2:95 (1977)) which
contains genes for ampicillin and tetracycline resistance. The
vector is digested with restriction enzyme and dephosphorylated.
The PCR amplified sequences are then ligated into the vector. The
vector will preferably include sequences which encode for an
antibiotic resistance gene, a trp promoter, a polyhis leader
(including the first six STII codons, polyhis sequence, and
enterokinase cleavage site), the PRO10282 or PRO19578 coding
region, lambda transcriptional terminator, and an argU gene.
[0272] The ligation mixture is then used to transform a selected E.
coli strain using the methods described in Sambrook et al., supra.
Transformants are identified by their ability to grow on LB plates
and antibiotic resistant colonies are then selected. Plasmid DNA
can be isolated and confirmed by restriction analysis and DNA
sequencing.
[0273] Selected clones can be grown overnight in liquid culture
medium such as LB broth supplemented with antibiotics. The
overnight culture may subsequently be used to inoculate a larger
scale culture. The cells are then grown to a desired optical
density, during which the expression promoter is turned on.
[0274] After culturing the cells for several more hours, the cells
can be harvested by centrifugation. The cell pellet obtained by the
centrifugation can be solubilized using various agents known in the
art, and the solubilized PRO10282 protein can then be purified
using a metal chelating column under conditions that allow tight
binding of the protein.
[0275] PRO10282 or PRO19578 may be expressed in E. coli in a
poly-His tagged form, using the following procedure. The DNA
encoding PRO10282 or PRO19578 is initially amplified using selected
PCR primers. The primers will contain restriction enzyme sites
which correspond to the restriction enzyme sites on the selected
expression vector, and other useful sequences providing for
efficient and reliable translation initiation, rapid purification
on a metal chelation column, and proteolytic removal with
enterokinase. The PCR-amplified, poly-His tagged sequences are then
ligated into an expression vector, which is used to transform an E.
coli host based on strain 52 (W3110 fuhA(tonA) lon galE
rpoHts(htpRts) clpP(lacIq). Transformants are first grown in LB
containing 50 mg/ml carbenicillin at 30.degree. C. with shaking
until an O.D.600 of 3-5 is reached. Cultures are then diluted
50-100 fold into CRA media (prepared by mixing 3.57 g
(NH.sub.4).sub.2SO.sub.4, 0.71 g sodium citrate2H2O, 1.07 g KCl,
5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500 mL
water, as well as 110 mM MPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM
MgSO.sub.4) and grown for approximately 20-30 hours at 30.degree.
C. with shaking. Samples are removed to verify expression by
SDS-PAGE analysis, and the bulk culture is centrifuged to pellet
the cells. Cell pellets are frozen until purification and
refolding.
[0276] E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets)
is resuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH
8 buffer. Solid sodium sulfite and sodium tetrathionate is added to
make final concentrations of 0.1 M and 0.02 M, respectively, and
the solution is stirred overnight at 4.degree. C. This step results
in a denatured protein with all cysteine residues blocked by
sulfitolization. The solution is centrifuged at 40,000 rpm in a
Beckman Ultracentrifuge for 30 min. The supernatant is diluted with
3-5 volumes of metal chelate column buffer (6 M guanidine, 20 mM
Tris, pH 7.4) and filtered through 0.22 micron filters to clarify.
The clarified extract is loaded onto a 5 ml Qiagen Ni-NTA metal
chelate column equilibrated in the metal chelate column buffer. The
column is washed with additional buffer containing 50 mM imidazole
(Calbiochem, Utrol grade), pH 7.4. The protein is eluted with
buffer containing 250 mM imidazole. Fractions containing the
desired protein are pooled and stored at 4.degree. C. Protein
concentration is estimated by its absorbance at 280 nm using the
calculated extinction coefficient based on its amino acid
sequence.
[0277] The proteins are refolded by diluting the sample slowly into
freshly prepared refolding buffer consisting of: 20 mM Tris, pH
8.6, 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM
EDTA. Refolding volumes are chosen so that the final protein
concentration is between 50 to 100 micrograms/ml. The refolding
solution is stirred gently at 4.degree. C. for 12-36 hours. The
refolding reaction is quenched by the addition of TFA to a final
concentration of 0.4% (pH of approximately 3). Before further
purification of the protein, the solution is filtered through a
0.22 micron filter and acetonitrile is added to 2-10% final
concentration. The refolded protein is chromatographed on a Poros
R1/H reversed phase column using a mobile buffer of 0.1% TFA with
elution with a gradient of acetonitrile from 10 to 80%. Aliquots of
fractions with A280 absorbance are analyzed on SDS polyacrylamide
gels and fractions containing homogeneous refolded protein are
pooled. Generally, the properly refolded species of most proteins
are eluted at the lowest concentrations of acetonitrile since those
species are the most compact with their hydrophobic interiors
shielded from interaction with the reversed phase resin. Aggregated
species are usually eluted at higher acetonitrile concentrations.
In addition to resolving misfolded forms of proteins from the
desired form, the reversed phase step also removes endotoxin from
the samples.
[0278] Fractions containing the desired folded PRO10282 or PRO19578
polypeptide are pooled and the acetonitrile removed using a gentle
stream of nitrogen directed at the solution. Proteins are
formulated into 20 mM Hepes, pH 6.8 with 0.14 M sodium chloride and
4% mannitol by dialysis or by gel filtration using G25 Superfine
(Pharmacia) resins equilibrated in the formulation buffer and
sterile filtered.
[0279] Specifically, two extracellular domains (ECD) of the native
human Stra6 protein PRO10282, Pepetide A (amino acids 229-295) and
Pepetide B (amino acids 532-667) were expressed separately as
peptides in the E. coli cytoplasm with an N-terminal polyhistidine
leader having the amino acid sequence MKHQHQHQHQHQHQMHQ (SEQ ID NO:
12). This leader provides for optimal translation initiation,
purification on a nickel chelation column, and efficient removal if
desired with the TAGZyme system (Unizyme Laboratories).
Transcription was controlled by the E. coli alkaline phosphatase
promoter (Kikuchi et al., Nucleic Acids Res. 9:5671-5678 [1981])
and the trp operon ribosome binding site (Yanofsky et al., Nucleic
Acids Res. 9:6647-6668 [1981]) provided for translation. Downstream
of the translation termination codon, is the .lambda.to
transcriptional terminator (Scholtissek and Grosse, Nucleic Acids
Res. 15:3185 [1987]) followed by the rare codon tRNA genes pro2,
argU, and glyT (Komine et al., J. Mol Biol. 212:579-598 [1990];
Fournier and Ozeki, Microbiol. Rev. 49:379-397 [1985]). The two
Stra6 ECD coding sequence DNA fragments were prepared by PCR from a
full length cDNA clone, and inserted into the expression vector
described above, which was designated as pST239. After DNA sequence
verification, the new Stra6 expression plasmids, designated
PE148380A and PE148380B, were transformed into the E. coli strain
58F3 ((fhuA.DELTA.(tonA.DELTA.) lon.DELTA. galE rpoHts (htpRts)
.DELTA.clpP laclq .DELTA.ompT.DELTA.(nmpc-fepE) .DELTA.slyD). Luria
Broth cultures of these transformants were first grown overnight at
30.degree. C., and then diluted 100 fold into a phosphate limiting
media to induce the alkaline phosphatase promoter. After 24 hours
at 30.degree. C. with shaking, the cultures were centrifuged, and
the cell pastes frozen until the start of peptide purification.
[0280] For purification, E. coli pastes (6-10 gm pellets) were
resuspended in 10 volumes (w/v) of 7 M guanidine HCl, 20 mM Tris,
pH 8, buffer. Solid sodium sulfite and sodium tetrathionate were
added to make final concentrations of 0.1 M and 0.02 M,
respectively, and the solution was stirred overnight at 4.degree.
C. The solution was clarified by centrifugation and loaded onto a
Qiagen Ni-NTA metal chelate column equilibrated in 6 M guanidine,
HCl, 20 mM Tris, pH 7.4. The column was washed with additional
buffer containing 50 mM imidazole (Calbiochem, Utrol grade). The
protein was eluted with buffer containing 250 mM imidazole.
Fractions containing the desired protein were pooled, dialyzed
against 1 mM HCl and stored at 4.degree. C.
Example 4
Expression of PRO10282 and PRO19578 in Mammalian Cells
[0281] This example illustrates preparation of a potentially
glycosylated form of PRO10282 and PRO19578 by recombinant
expression in mammalian cells.
[0282] The vector, pRK5 (see EP 307,247, published Mar. 15, 1989),
is employed as the expression vector. Optionally, the PRO10282 or
PRO19578 DNA is ligated into pRK5 with selected restriction enzymes
to allow insertion of the PRO10282 DNA using ligation methods such
as described in Sambrook et al., supra. The resulting vector is
called pRK5-PRO10282 and pRK5-PRO19578, respectively.
[0283] In one embodiment, the selected host cells may be 293 cells.
Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue
culture plates in medium such as DMEM supplemented with fetal calf
serum and optionally, nutrient components and/or antibiotics. About
10 .mu.g pRK5-PRO10282 or pRK5-PRO19578 DNA is mixed with about 1
.mu.g DNA encoding the VA RNA gene [Thimmappaya et al., Cell,
31:543 (1982)] and dissolved in 500 .mu.l of 1 mM Tris-HCl, 0.1 mM
EDTA, 0.227 M CaCl.sub.2. To this mixture is added, dropwise, 500
.mu.l of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO.sub.4, and
a precipitate is allowed to form for 10 minutes at 25.degree. C.
The precipitate is suspended and added to the 293 cells and allowed
to settle for about four hours at 37.degree. C. The culture medium
is aspirated off and 2 ml of 20% glycerol in PBS is added for 30
seconds. The 293 cells are then washed with serum free medium,
fresh medium is added and the cells are incubated for about 5
days.
[0284] Approximately 24 hours after the transfections, the culture
medium is removed and replaced with culture medium (alone) or
culture medium containing 200 .mu.Ci/ml .sup.35S-cysteine and 200
.mu.Ci/ml .sup.35S-methionine. After a 12 hour incubation, the
conditioned medium is collected, concentrated on a spin filter, and
loaded onto a 15% SDS gel. The processed gel may be dried and
exposed to film for a selected period of time to reveal the
presence of PRO10282 polypeptide. The cultures containing
transfected cells may undergo further incubation (in serum free
medium) and the medium is tested in selected bioassays.
[0285] In an alternative technique, PRO10282 or PRO19578 may be
introduced into 293 cells transiently using the dextran sulfate
method described by Somparyrac et al., Proc. Natl. Acad. Sci.,
12:7575 (1981). 293 cells are grown to maximal density in a spinner
flask and 700 .mu.g pRK5-PRO10282 or pRK5-PRO19578 DNA is added.
The cells are first concentrated from the spinner flask by
centrifugation and washed with PBS. The DNA-dextran precipitate is
incubated on the cell pellet for four hours. The cells are treated
with 20% glycerol for 90 seconds, washed with tissue culture
medium, and re-introduced into the spinner flask containing tissue
culture medium, 5 .mu.g/ml bovine insulin and 0.1 .mu.g/ml bovine
transferrin. After about four days, the conditioned media is
centrifuged and filtered to remove cells and debris. The sample
containing expressed PRO10282 or PRO19578 can then be concentrated
and purified by any selected method, such as dialysis and/or column
chromatography.
[0286] In another embodiment, PRO10282 or PRO19578 can be expressed
in CHO cells. The pRK5-PRO10282 or pRK5-PRO19578 can be transfected
into CHO cells using known reagents such as CaPO.sub.4 or
DEAE-dextran. As described above, the cell cultures can be
incubated, and the medium replaced with culture medium (alone) or
medium containing a radiolabel such as .sup.35S-methionine. After
determining the presence of PRO10282 or PRO19578 polypeptide, the
culture medium may be replaced with serum free medium. Preferably,
the cultures are incubated for about 6 days, and then the
conditioned medium is harvested. The medium containing the
expressed PRO10282 or PRO19578 can then be concentrated and
purified by any selected method.
[0287] Epitope-tagged PRO10282 or pRO19578 may also be expressed in
host CHO cells. The PRO10282 or PRO19578 may be subcloned out of
the pRK5 vector. The subclone insert can undergo PCR to fuse in
frame with a selected epitope tag such as a poly-his tag into a
Baculovirus expression vector. The poly-his tagged PRO10282 or
PRO19578 insert can then be subcloned into a SV40 driven vector
containing a selection marker such as DHFR for selection of stable
clones. Finally, the CHO cells can be transfected (as described
above) with the SV40 driven vector. Labeling may be performed, as
described above, to verify expression. The culture medium
containing the expressed poly-His tagged PRO10282 or PRO19578 can
then be concentrated and purified by any selected method, such as
by Ni.sup.2+-chelate affinity chromatography.
[0288] PRO10282 or PRO19578 may also be expressed in CHO and/or COS
cells by a transient expression procedure or in CHO cells by
another stable expression procedure.
[0289] Stable expression in CHO cells is performed using the
following procedure. The proteins are expressed as an IgG construct
(immunoadhesin), in which the coding sequences for the soluble
forms (e.g. extracellular domains) of the respective proteins are
fused to an IgG1 constant region sequence containing the hinge, CH2
and CH2 domains and/or is a poly-His tagged form.
[0290] Following PCR amplification, the respective DNAs are
subcloned in a CHO expression vector using standard techniques as
described in Ausubel et al., Current Protocols of Molecular
Biology, Unit 3.16, John Wiley and Sons (1997). CHO expression
vectors are constructed to have compatible restriction sites 5' and
3' of the DNA of interest to allow the convenient shuttling of
cDNA's. The vector used expression in CHO cells is as described in
Lucas et al., Nucl. Acids Res. 24:9 (1774-1779 (1996), and uses the
SV40 early promoter/enhancer to drive expression of the cDNA of
interest and dihydrofolate reductase (DHFR). DHFR expression
permits selection for stable maintenance of the plasmid following
transfection.
[0291] Twelve micrograms of the desired plasmid DNA is introduced
into approximately 10 million CHO cells using commercially
available transfection reagents Superfect.RTM. (Quiagen),
Dosper.RTM. or Fugene.RTM. (Boebringer Mannheim). The cells are
grown as described in Lucas et al., supra. Approximately
3.times.10.sup.-7 cells are frozen in an ampoule for further growth
and production as described below.
[0292] The ampoules containing the plasmid DNA are thawed by
placement into water bath and mixed by vortexing. The contents are
pipetted into a centrifuge tube containing 10 mLs of media and
centrifuged at 1000 rpm for 5 minutes. The supernatant is aspirated
and the cells are resuspended in 10 mL of selective media (0.2
.mu.m filtered PS20 with 5% 0.2 .mu.m diafiltered fetal bovine
serum). The cells are then aliquoted into a 100 mL spinner
containing 90 mL of selective media. After 1-2 days, the cells are
transferred into a 250 mL spinner filled with 150 mL selective
growth medium and incubated at 37.degree. C. After another 2-3
days, 250 mL, 500 mL and 2000 mL spinners are seeded with
3.times.10.sup.5 The cell media is exchanged with fresh media by
centrifugation and resuspension in production medium. Although any
suitable CHO media may be employed, a production medium described
in U.S. Pat. No. 5,122,469, issued Jun. 16, 1992 may actually be
used. A 3L production spinner is seeded at 1.2.times.10.sup.6
cells/mL. On day 0, the cell number pH is determined. On day 1, the
spinner is sampled and sparging with filtered air is commenced. On
day 2, the spinner is sampled, the temperature shifted to
33.degree. C., and 30 mL of 500 g/L glucose and 0.6 mL of 10%
antifoam (e.g., 35% polydimethylsiloxane emulsion, Dow Coming 365
Medical Grade Emulsion) taken. Throughout the production, the pH is
adjusted as necessary to keep it at around 7.2. After 10 days, or
until the viability dropped below 70%, the cell culture is
harvested by centrifugation and filtering through a 0.22 .mu.m
filter. The filtrate was either stored at 4.degree. C. or
immediately loaded onto columns for purification.
[0293] For the poly-His tagged constructs, the proteins are
purified using a Ni-NTA column (Qiagen). Before purification,
imidazole is added to the conditioned media to a concentration of 5
mM. The conditioned media is pumped onto a 6 ml Ni-NTA column
equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl
and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4.degree. C.
After loading, the column is washed with additional equilibration
buffer and the protein eluted with equilibration buffer containing
0.25 M imidazole. The highly purified protein is subsequently
desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl
and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia)
column and stored at -80.degree. C.
[0294] Immunoadhesin (Fc-containing) constructs are purified from
the conditioned media as follows. The conditioned medium is pumped
onto a 5 ml Protein A column (Pharmacia) which had been
equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading,
the column is washed extensively with equilibration buffer before
elution with 100 mM citric acid, pH 3.5. The eluted protein is
immediately neutralized by collecting 1 ml fractions into tubes
containing 275 .mu.L of 1 M Tris buffer, pH 9. The highly purified
protein is subsequently desalted into storage buffer as described
above for the poly-His tagged proteins. The homogeneity is assessed
by SDS polyacrylamide gels and by N-terminal amino acid sequencing
by Edman degradation.
Example 5
Expression of PRO10282 and PRO19578 in Yeast
[0295] The following method describes recombinant expression of
PRO10282 and PRO19578 in yeast.
[0296] First, yeast expression vectors are constructed for
intracellular production or secretion of PRO10282 polypeptides from
the ADH2/GAPDH promoter. DNA encoding a PRO10282 polypeptide
(including PRO19578) and the promoter is inserted into suitable
restriction enzyme sites in the selected plasmid to direct
intracellular expression of PRO10282. For secretion, DNA encoding
PRO10282 can be cloned into the selected plasmid, together with DNA
encoding the ADH2/GAPDH promoter, a native PRO10282 signal peptide
or other mammalian signal peptide, or, for example, a yeast
alpha-factor or invertase secretory signal/leader sequence, and
linker sequences (if needed) for expression of PRO10282.
[0297] Yeast cells, such as yeast strain AB110, can then be
transformed with the expression plasmids described above and
cultured in selected fermentation media. The transformed yeast
supernatants can be analyzed by precipitation with 10%
trichloroacetic acid and separation by SDS-PAGE, followed by
staining of the gels with Coomassie Blue stain.
[0298] Recombinant PRO10282 (including PRO19578) can subsequently
be isolated and purified by removing the yeast cells from the
fermentation medium by centrifugation and then concentrating the
medium using selected cartridge filters. The concentrate containing
PRO10282 (e.g. PRO19578) may further be purified using selected
column chromatography resins.
Example 6
Expression of PRO10282 and PRO19578 in Baculovirus-Infected Insect
Cells
[0299] The following method describes recombinant expression of
PRO10282 and PRO19578 in Baculovirus-Infected Insect Cells.
[0300] The sequence coding for PRO10282 or PRO19578 is fused
upstream of an epitope tag contained within a baculovirus
expression vector. Such epitope tags include poly-his tags and
immunoglobulin tags (like Fc regions of IgG). A variety of plasmids
may be employed, including plasmids derived from commercially
available plasmids such as pVL1393 (Novagen). Briefly, the sequence
encoding PRO10282 or PRO19578 or the desired portion of the coding
sequence of PRO10282 or PRO19578, such as the sequence encoding the
extracellular domain of a transmembrane protein or the sequence
encoding the mature protein if the protein is extracellular, is
amplified by PCR with primers complementary to the 5' and 3'
regions. The 5' primer may incorporate flanking (selected)
restriction enzyme sites. The product is then digested with those
selected restriction enzymes and subcloned into the expression
vector.
[0301] Recombinant baculovirus is generated by co-transfecting the
above plasmid and BaculoGold.TM. virus DNA (Pharmingen) into
Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711) using
lipofectin (commercially available from GIBCO-BRL). After 4-5 days
of incubation at 28.degree. C., the released viruses are harvested
and used for further amplifications. Viral infection and protein
expression are performed as described by O'Reilley et al.,
Baculovirus expression vectors: A Laboratory Manual, Oxford: Oxford
University Press (1994).
[0302] Expressed poly-his tagged PRO10282 or PRO19578 can then be
purified, for example, by Ni.sup.2+-chelate affinity chromatography
as follows. Extracts are prepared from recombinant virus-infected
Sf9 cells as described by Rupert et al., Nature 362:175-179 (1993).
Briefly, Sf9 cells are washed, resuspended in sonication buffer (25
mL Hepes, pH 7.9; 12.5 mM MgCl.sub.2; 0.1 mM EDTA; 10% glycerol;
0.1% NP-40; 0.4 M KCl), and sonicated twice for 20 seconds on ice.
The sonicates are cleared by centrifugation, and the supernatant is
diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl,
10% glycerol, pH 7.8) and filtered through a 0.45 .mu.m filter. A
Ni.sup.2+-NTA agarose column (commercially available from Qiagen)
is prepared with a bed volume of 5 mL, washed with 25 mL of water
and equilibrated with 25 mL of loading buffer. The filtered cell
extract is loaded onto the column at 0.5 mL per minute. The column
is washed to baseline A.sub.280 with loading buffer, at which point
fraction collection is started. Next, the column is washed with a
secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol,
pH 6.0), which elutes nonspecifically bound protein. After reaching
A.sub.280 baseline again, the column is developed with a 0 to 500
mM Imidazole gradient in the secondary wash buffer. One mL
fractions are collected and analyzed by SDS-PAGE and silver
staining or Western blot with Ni.sup.2+-NTA-conjugated to alkaline
phosphatase (Qiagen). Fractions containing the eluted
His.sub.10-tagged PRO10282 or PRO19578 are pooled and dialyzed
against loading buffer.
[0303] Alternatively, purification of the IgG tagged (or Fc tagged)
PRO10282 or PRO19578 can be performed using known chromatography
techniques, including for instance, Protein A or protein G column
chromatography.
Example 7
Preparation of Antibodies that Bind PRO10282 or PRO19578
[0304] This example illustrates preparation of monoclonal
antibodies which can specifically bind PRO10282 or PRO19578.
[0305] Techniques for producing the monoclonal antibodies are known
in the art and are described, for instance, in Goding, supra.
Immunogens that may be employed include purified PRO10282 and
PRO19578, fusion proteins containing PRO10282 or PRO19578, and
cells expressing recombinant PRO10282 or PRO19578 on the cell
surface. Selection of the immunogen can be made by the skilled
artisan without undue experimentation.
[0306] Mice, such as Balb/c, are immunized with the PRO10282 or
PRO19578 immunogen emulsified in complete Freund's adjuvant and
injected subcutaneously or intraperitoneally in an amount from
1-100 micrograms. Alternatively, the immunogen is emulsified in
MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, Mont.)
and injected into the animal's hind foot pads. The immunized mice
are then boosted 10 to 12 days later with additional immunogen
emulsified in the selected adjuvant. Thereafter, for several weeks,
the mice may also be boosted with additional immunization
injections. Serum samples may be periodically obtained from the
mice by retro-orbital bleeding for testing in ELISA assays to
detect anti-PRO 10282 antibodies or anti-PRO 19578 antibodies.
[0307] After a suitable antibody titer has been detected, the
animals "positive" for antibodies can be injected with a final
intravenous injection of PRO10282 or PRO19578. Three to four days
later, the mice are sacrificed and the spleen cells are harvested.
The spleen cells are then fused (using 35% polyethylene glycol) to
a selected murine myeloma cell line such as P3X63AgU.1, available
from ATCC, No. CRL 1597. The fusions generate hybridoma cells which
can then be plated in 96 well tissue culture plates containing HAT
(hypoxanthine, aminopterin, and thymidine) medium to inhibit
proliferation of non-fused cells, myeloma hybrids, and spleen cell
hybrids.
[0308] The hybridoma cells will be screened in an ELISA for
reactivity against PRO10282 or PRO19578. Determination of
"positive" hybridoma cells secreting the desired monoclonal
antibodies against PRO10282 or PRO19578 is within the skill in the
art.
[0309] The positive hybridoma cells can be injected
intraperitoneally into syngeneic Balb/c mice to produce ascites
containing the anti-PRO10282 or anti-PRO19578 monoclonal
antibodies. Alternatively, the hybridoma cells can be grown in
tissue culture flasks or roller bottles. Purification of the
monoclonal antibodies produced in the ascites can be accomplished
using ammonium sulfate precipitation, followed by gel exclusion
chromatography. Alternatively, affinity chromatography based upon
binding of antibody to protein A or protein G can be employed.
[0310] Specifically, five Balb/c mice (Charles River Laboratories,
Wilmington, Del.) were hyper-immunized with purified
Unizyme-conjugated amino acid peptide, corresponding to amino acids
532-667 of human Stra6, in Ribi adjuvant (Ribi Immunochem Research,
Inc., Hamilton, Mont.). B-cells from popliteal lymph nodes were
fused with mouse myeloma cells (X63.Ag8.653; American Type Culture
Collection, Rockville, Md.) as previously described (Hongo et al.,
Hybridoma 14:253-260 [1995]). After 10-14 days, supernatants were
harvested and screened for antibody production by direct
enzyme-linked immunosorbant assay (ELISA). Eight positive clones,
showing the highest immunobinding by direct ELISA and
immunohistochemistry after two rounds of subcloning by limiting
dilution, were injected into Pristine-primed mice for in vivo
production of miAb (Freud and Blair, J. Immunol 129:2826-2830
[1982]). The ascites fluids were pooled and purified by Protein A
affinity chromatography (Pharmacia fast protein liquid
chromatography (FPLC); Pharmacia, Uppsala, Sweden) as previously
described (Hongo et al., supra). The purified antibody preparations
were sterile filtered (0.2-.mu.m pore size; Nalgene, Rochester,
N.Y.) and stored at 4.degree. C. in phosphate buffered saline
(PBS).
Example 8
Purification of PRO10282 and PRO19578 Polypeptides Using Specific
Antibodies
[0311] Native or recombinant PRO10282 and PRO19578 polypeptides may
be purified by a variety of standard techniques in the art of
protein purification. For example, pro-PRO10282 or pro-PRO19578
polypeptide, mature PRO10282 or PRO19578 polypeptide, or
pre-PRO10282 or pre-PRO19578 polypeptide is purified by
immunoaffinity chromatography using antibodies specific for the
PRO10282 polypeptide of interest. In general, an immunoaffinity
column is constructed by covalently coupling the anti-PRO10282 or
anti-PRO19578 polypeptide antibody to an activated chromatographic
resin.
[0312] Polyclonal immunoglobulins are prepared from immune sera
either by precipitation with ammonium sulfate or by purification on
immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway,
N.J.). Likewise, monoclonal antibodies are prepared from mouse
ascites fluid by ammonium sulfate precipitation or chromatography
on immobilized Protein A. Partially purified immunoglobulin is
covalently attached to a chromatographic resin such as
CnBr-activated SEPHAROSE.TM. (Pharmacia LKB Biotechnology). The
antibody is coupled to the resin, the resin is blocked, and the
derivative resin is washed according to the manufacturer's
instructions.
[0313] Such an immunoaffinity column is utilized in the
purification of PRO10282 or PRO19578 polypeptide by preparing a
fraction from cells containing PRO10282 or PRO19578 polypeptide in
a soluble form. This preparation is derived by solubilization of
the whole cell or of a subcellular fraction obtained via
differential centrifugation by the addition of detergent or by
other methods well known in the art. Alternatively, soluble
PRO10282 or PRO19578 polypeptide containing a signal sequence may
be secreted in useful quantity into the medium in which the cells
are grown.
[0314] A soluble PRO10282 or PRO19578 polypeptide-containing
preparation is passed over the immunoaffinity column, and the
column is washed under conditions that allow the preferential
absorbance of PRO10282 or PRO19578 polypeptide (e.g., high ionic
strength buffers in the presence of detergent). Then, the column is
eluted under conditions that disrupt antibody/PRO10282 or PRO19578
polypeptide binding (e.g., a low pH buffer such as approximately pH
2-3, or a high concentration of a chaotrope such as urea or
thiocyanate ion), and PRO10282 or pRO19578 polypeptide is
collected.
Example 9
Drug Screening
[0315] This invention is particularly useful for screening
compounds by using PRO10282 (Stra6) polypeptides (including
PRO19578) or binding fragment thereof in any of a variety of drug
screening techniques. The PRO10282 polypeptide or fragment employed
in such a test may either be free in solution, affixed to a solid
support, borne on a cell surface, or located intracellularly. One
method of drug screening utilizes eukaryotic or prokaryotic host
cells which are stably transformed with recombinant nucleic acids
expressing the PRO10282 polypeptide or fragment. Drugs are screened
against such transformed cells in competitive binding assays. Such
cells, either in viable or fixed form, can be used for standard
binding assays. One may measure, for example, the formation of
complexes between PRO10282 polypeptide or a fragment and the agent
being tested. Alternatively, one can examine the diminution in
complex formation between the PRO10282 polypeptide and its target
cell or target receptors caused by the agent being tested.
[0316] Thus, the present invention provides methods of screening
for drugs or any other agents which can affect a PRO10282 (Stra6)
polypeptide-associated disease or disorder. These methods comprise
contacting such an agent with a Stra6 polypeptide or fragment
thereof and assaying (i) for the presence of a complex between the
agent and the Stra6 polypeptide or fragment, or (ii) for the
presence of a complex between the Stra6 polypeptide or fragment and
the cell, by methods well known in the art. In such competitive
binding assays, the Stra6 polypeptide or fragment is typically
labeled. After suitable incubation, free Stra6 polypeptide or
fragment is separated from that present in bound form, and the
amount of free or uncomplexed label is a measure of the ability of
the particular agent to bind to Stra6 polypeptide or to interfere
with the Stra6 polypeptide/cell complex.
[0317] Another technique for drug screening provides high
throughput screening for compounds having suitable binding affinity
to a polypeptide and is described in detail in WO 84/03564,
published on Sep. 13, 1984. Briefly stated, large numbers of
different small peptide test compounds are synthesized on a solid
substrate, such as plastic pins or some other surface. As applied
to a PRO10282 (Stra6) polypeptide, the peptide test compounds are
reacted with Stra6 polypeptide and washed. Bound Stra6 polypeptide
is detected by methods well known in the art. Purified Stra6
polypeptide can also be coated directly onto plates for use in the
aforementioned drug screening techniques. In addition,
non-neutralizing antibodies can be used to capture the peptide and
immobilize it on the solid support.
[0318] This invention also contemplates the use of competitive drug
screening assays in which neutralizing antibodies capable of
binding a Stra6 polypeptide specifically compete with a test
compound for binding to a Stra6 polypeptide or fragments thereof.
In this manner, the antibodies can be used to detect the presence
of any peptide which shares one or more antigenic determinants with
the Stra6 polypeptide.
Example 10
Rational Drug Design
[0319] The goal of rational drug design is to produce structural
analogs of biologically active polypeptide of interest (i.e., a
PRO10282 (Stra6) polypeptide) or of small molecules with which they
interact, e.g., agonists, antagonists, or inhibitors. Any of these
examples can be used to fashion drugs which are more active or
stable forms of the native sequence PRO10282 polypeptide or
PRO19578 polypeptide, or which enhance or interfere with the
function of the native sequence PRO10282 or PRO19578 polypeptide in
vivo (cf:, Hodgson, Bio/Technology, 9: 19-21 (1991)).
[0320] In one approach, the three-dimensional structure of the
native sequence PRO10282 or PRO19578 polypeptide, or of a PRO10282
or PRO19578 polypeptide-inhibitor complex, is determined by x-ray
crystallography, by computer modeling or, most typically, by a
combination of the two approaches. Both the shape and charges of
the native PRO10282 or PRO19578 polypeptide must be ascertained to
elucidate the structure and to determine active site(s) of the
molecule. Less often, useful information regarding the structure of
the PRO10282 or PRO19578 polypeptide may be gained by modeling
based on the structure of homologous proteins. In both cases,
relevant structural information is used to design analogous
PRO10282 polypeptide-like molecules or to identify efficient
inhibitors. Useful examples of rational drug design may include
molecules which have improved activity or stability as shown by
Braxton and Wells, Biochemistry 31:7796-7801 (1992) or which act as
inhibitors, agonists, or antagonists of native peptides as shown by
Athauda et al., J. Biochem., 113:742-746 (1993).
[0321] It is also possible to isolate a target-specific antibody,
selected by functional assay, as described above, and then to solve
its crystal structure. This approach, in principle, yields a
pharmacore upon which subsequent drug design can be based. It is
possible to bypass protein crystallography altogether by generating
anti-idiotypic antibodies (anti-ids) to a functional,
pharmacologically active antibody. As a mirror image of a mirror
image, the binding site of the anti-ids would be expected to be an
analog of the original receptor. The anti-id could then be used to
identify and isolate peptides from banks of chemically or
biologically produced peptides. The isolated peptides would then
act as the pharmacore.
[0322] By virtue of the present invention, sufficient amounts of
the PRO10282 polypeptides (including PRO19578) may be made
available to perform such analytical studies as X-ray
crystallography. In addition, knowledge of the PRO10282 polypeptide
amino acid sequences provided herein will provide guidance to those
employing computer modeling techniques in place of or in addition
to x-ray crystallography.
Example 11
Tissue Expression Distribution
[0323] Oligonucleotide probes were constructed from the PRO10282
polypeptide-encoding nucleotide sequence shown in the accompanying
figures for use in quantitative PCR amplification reactions. The
oligonucleotide probes were chosen so as to give an approximately
200-600 base pair amplified fragment from the 3' end of its
associated template in a standard PCR reaction. The oligonucleotide
probes were employed in standard quantitative PCR amplification
reactions with Clontech RNA isolated from different adult human
tissue sources and analyzed by agarose gel electrophoresis so as to
obtain a quantitative determination of the level of expression of
the PRO10282 polypeptide-encoding nucleic acid in the various
tissues tested. Knowledge of the expression pattern or the
differential expression of the PRO10282 polypeptide-encoding
nucleic acid in various different human tissue types provides a
diagnostic marker useful for tissue typing, with or without other
tissue-specific markers, for determining the primary tissue source
of a metastatic tumor, and the like. The results of these assays
(shown in FIG. 10) demonstrated that the DNA148380-2827 molecule is
highly expressed in the adult kidney, testis and uterus;
significantly expressed in breast, prostate and trachea; weakly
expressed in brain, heart, lung and thymus; and not expressed in
liver, bone marrow, colon, skeletal muscle, small intestine, spleen
and stomach.
[0324] Total RNA was purchased from Clontech (Palo Alto, Calif.)
and analyzed using the following primer/probe set for PCR
amplification:
1 h.Stra6.tmf3: 5' CACACTCGAGAGCCAGATATTTT (SEQ ID NO: 6)
h.Stra5.tmr4: 5' AACAAGTTTATTGCAGGGAACAC (SEQ ID NO: 7)
h.Stra6.tmp4: 5' TGTAGTTTTTATGCCTTTGGCTATTATGAAAGAGGT (SEQ ID NO:
8) tmf = forward primer tmr = reverse primer tmp = probe
[0325] In situ hybridization, performed as described in Example 12
below, confirmed Stra6 expression in kidney tubular epithelial
cells, myometrium, and stromal cells surrounding breast ducts and
lobules, whereas little or no expression was detected in sections
of brain, liver, spleen, pancreas, heart, lung, stomach, small
intestine, colon, prostate, spleen, and adrenal cortex (data not
shown). Of the normal tissues examined by in situ hybridization,
highest expression levels were seen in placenta and adrenal
medulla, which were not included in the PCR analysis.
Example 12
Over-expression of Native Human PRO10282 (Stra6) Transcript in
Human Tumors
[0326] This example shows that the gene encoding native human
full-length PRO10282 (Stra6) is significantly over-expressed in
certain human colon tumors and also in cell lines derived from
various human tumors such as colon, lung, kidney and breast.
[0327] The starting material for the screen was total RNA isolated
from human colon tumors, or various human colon, kidney, breast, or
lung tumor cell lines. In colon tumor tissue, Stra6 RNA expression
was determined relative to RNA from normal colon tissue (mucosa)
from the same patient. Stra6 RNA expression in various tumor cell
lines was determined in comparison with various normal cell lines
(i.e., normal colon, kidney and lung cell lines).
[0328] Real-time quantitative PCR (RT-PCR, for example, TAQMAN ABI
PRIZM 7700.TM. Sequence Detection System.TM. [Perkin Elmer, Applied
Biosystems Division, Foster City, Calif.]), was used to monitor
quantitative differences in the level of expression of the PRO10292
(Stra6) encoding gene (corresponding to DNA148380-2827) in normal
cells and cells derived from certain cancers or cancer cell lines,
using Taqman assay reagents. 50 .mu.l RT-PCR reactions consisted of
5 .mu.l 10.times. Taqman Buffer A, 300 .mu.M of each dNTF, 5 mM
MgCl.sub.2, 10 units of RNAse inhibitor, 12.5 units of MuLV Reverse
Transcriptase, 1.25 units of AmpliTaq Gold DNA Polymerase, 200 nM
probe, 500 nM primers and 100 ng RNA. Reaction conditions consisted
of reverse transcription at 48.degree. C. for 30 minutes,
denaturation at 95.degree. C. for 25 seconds and 65.degree. C. for
one minute. Reaction products were analyzed on 4-20% polyacrylamide
gels (Novex).
[0329] Standard curves were used to determine relative levels of
expression for each gene of interest as well as the
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene
for each sample analyzed. Relative normalized units were obtained
by dividing the gene of interest mRNA level by the GAPDH mRNA
level. Relative normalized units were compared between experimental
sample and control to determine fold induction.
[0330] The results were used to determine whether the mRNA encoding
PRO10282 is over-expressed in any of the primary colon cancers or
colon, kidney, breast, or lung cancer cell lines that were
screened. The histology of some matched human normal and colon
tumor samples used for the analysis (see FIGS. 11 and 12) is shown
below:
2 Number Histology 850 Invasive adenocarcinoma; no necrosis; good
preservation. 851 Invasive adenocarcinoma; minimal necrosis; good
condition. 892 Invasive adenocarcinoma; minimal necrosis; good
condition. 869 Invasive adenocarcinoma; minimal necrosis; good
condition. 893 Normal mucosa - dysplasia - invasive adenocarcinoma;
minor necrosis; good condition. 870 Adenocarcinoma - severe
dysplasia; minimal necrosis; good condition. 871 Adenocarcinoma -
dysplasia - normal mucosa; no necrosis; good condition. 848
Adenocarcinoma - appears to be arising in villous adenoma; normal
mucosa/submucosa; no necrosis, good condition. 872 Invasive
adenocarcinoma; about 70% of tumor is necrotic; overall good
preservation. 778 Adenocarcinoma - unusually papillary morphology;
normal/hyperplastic mucosa; minimal necrosis; acceptable
preservation. 17 Moderately well-differentiated adenocarcmoma. 18
Well-differentiated adenocarcinoma. 64 Moderately
well-differentiated adenocarcinoma. 76 Moderately
well-differentiated adenocarcinoma.
[0331] Human lung cell lines include the normal human lung
fibroblast cell lines MRC5 (CCL-171) and IMR90 (CCL-186), the human
lung carcinoma epithelial cell line A549 (SRCC768, CCL-185), the
human epidermoid lung carcinoma cell line Calu-1 (SRCC769; HTB-54),
the human anaplastic carcinoma cell line Calu-6 (SRCC770,
HTB-56--probably lung), the human epithelial cell line NCI-H441
(SRCC772; HTB-174) which was derived from pericardial fluid of a
patient with papillary adenocarcinoma of the lung, and the human
lung squamous cell carcinoma cell line SW900 (SRCC775; HTB-59), all
available from ATCC.
[0332] Colon cell lines include, for example, the normal colon
fibroblast cell line CCD112Co (CRL-1541), the human colorectal
adenocarcinoma cell line CaCo-2 (HTB-37), the human colorectal
adenocarcinoma cell line WiDr (CCL-218), the human colorectal
carcinoma cell line HCT116 (CCL-247), the human colorectal
adenocarcinoma cell line SK-Col (HTB-39) , the human colorectal
adenocarcinoma cell line COLO320 (SRCC778; CCL-220), the human
colorectal adenocarcinoma cell line HT29 (SRCC779; HTB-38), the
human colorectal adenocarcinoma cell line SW403 (CCL-230), the
human colon cancer cell line NCI/HCC2998, and the human colorectal
adenocarcinoma cell line Colo320DM (CCL-220), all available from
ATCC or other public sources.
[0333] Human breast carcinoma cell lines include the human breast
adenocarcinoma cell line MCF7 (SRCC766; HTB-22), and the human
breast cancer cell line NCI/ADR-RES, both of which are publicly
available.
[0334] Kidney lines include the 293 cell line (CRL-1573) which is
transformed with adenovirus 5 DNA. Two Wilm's tumor cell lines were
also included in the analysis, G401 (CRL-1441) and SK-NEP-1
(CRL-1573).
[0335] RNA Preparation
[0336] RNA was prepared from the foregoing cultured cell lines. The
isolation was performed using purification kit, buffer set and
protease from Qiagen, according to the manufacturer's instructions
and the description below. More specifically, total RNA from cells
in culture was isolated using Qiagen RNeasy midi-columns. Total RNA
from tissue samples was isolated using RNA Stat-60 (Tel-Test). RNA
prepared from tumor was isolated by cesium chloride density
gradient centrifugation.
[0337] Solid Human Tumor Sample Preparation and Lysis
[0338] Tumor samples were weighed and placed into 50 ml conical
tubes and held on ice. Processing was limited to no more than 250
mg tissue per preparation (1 tip/preparation). The protease
solution was freshly prepared by diluting into 6.25 ml cold
ddH.sub.2O to a final concentration of 20 .mu.g/ml and stored at
4.degree. C. G2 buffer (20 ml) was prepared by diluting DNAse A to
a final concentration of 200 .mu.g/ml (from 100 mg/ml stock). The
tumor tissue was homogenized in 10 ml G2 buffer for 60 seconds
using the large tip of the polytron in a laminar-flow TC hood in
order to avoid inhalation of aerosols, and held at room
temperature. Between samples, the polytron was cleaned by spinning
at 2.times.30 seconds each in 2 L, ddH.sub.2O, followed by G2
buffer (50 ml). If tissue was still present on the generator tip,
the apparatus was disassembled and cleaned.
[0339] Qiagen protease (prepared as indicated above, 1.0 ml) was
added, followed by vortexing and incubation at 50.degree. C. for 3
hours. The incubation and centrifugation were repeated until the
lysates were clear (e.g., incubating additional 30-60 minutes,
pelleting at 3000.times.g for 10 minutes, 4.degree. C.).
[0340] Quantitation
[0341] The results obtained from the real-time PCR analysis of RNA
were initially expressed as delta CT units. One unit corresponds to
one PCR cycle or approximately a 2-fold amplification relative to
normal, two units correspond to 4-fold, 3 units to 8-fold
amplification and so on. The data is converted to fold difference
and presented as such. Initially, reverse transcriptase was used to
synthesize cDNA from 100 ng total RNA or polyA+RNA using oligo(dT)
as a primer. The resultant cDNA was then used as a template for
PCR. Qantitation was obtained using primers derived from the
3'-untranslated regions of the PRO10282 encoding sequence and a
TAQMAN.TM. fluorescent probe corresponding to the respective
intervening sequences. Using the 3' region tends to avoid crossing
intron-exon boundaries in the genomic DNA, an essential requirement
for accurate assessment of RNA expression using this method. The
sequences for the primers and probes (forward, reverse, and probe)
using for the PRO10282 encoding gene amplification were as
follows:
[0342] One set included the forward and reverse primers and probe
described in Example 11 above as SEQ ID Nos: 6, 7 and 8,
respectively.
3 Another set included: hStra6.tmfl1: 5'AGACCAGGTCCCACACTGA (SEQ ID
NO: 9) hStra6.tmr1: 5'TTCATAATAGCCAAAGGCATAAAA (SEQ ID NO: 10), and
h.Stra6.tmp1: 5'CTGCCCACAGTCGAGAGCCAGAT 3' (SEQ ID NO: 11) Human
GAPDH: forward primer: 5'-GAAGATGGTGATGGGAATTC-3' (SEQ ID NO: 14)
reverse primer; 5'-GAAGGTGAAGGTCGGAGTC-3' (SEQ ID NO: 15) probe:
5'-CAAGCTTCCCGTTCTCAGCC-3' (SEQ ID NO: 16)
[0343] The 5' nuclease assay reaction is a fluorescent PCR-based
technique which makes use of the 5' exonuclease activity of Taq DNA
polymerase enzyme to monitor amplification in real time. Two
oligonucleotide primers are used to generate an amplicon typical of
a PCR reaction. A third oligonucleotide, or probe, is designed to
detect nucleotide sequence located between the two PCR primers. The
probe is non-extendible by Taq DNA polymerase enzyme, and is
labeled with a reporter fluorescent dye and a quencher fluorescent
dye. Any laser-induced emission from the reporter dye is quenched
by the quenching dye when the two dyes are located close together
as they are on the probe. During the amplification reaction, the
Taq DNA polymerase enzyme cleaves the probe in a template-dependent
manner. The resultant probe fragments disassociate in solution, and
signal from the released reporter dye is free from the quenching
effect of the second fluorophore. One molecule of reporter dye is
liberated for each new molecule synthesized, and detection of the
unquenched reporter dye provides the basis for quantitative
interpretation of the data.
[0344] As noted above, the 5' nuclease procedure is run on a
real-time quantitative PCR device such as the ABI PRIZM
.sub.7700.TM. Sequence Detection System.TM.. The system consists of
a thermocyler, laser, charge-coupled device (CCD), camera and
computer. The system amplifies samples in a 96-well format on a
thermocycler. During amplification, laser-induced fluorescent
signal is collected in real-time through fiber optics cables for
all 96 wells, and detected at the CCD. The system includes software
for running the instrument and for analyzing the data.
[0345] 5'-Nuclease assay data are initially expressed as Ct, or the
threshold cycle. This is defined as the cycle at which the reporter
signal accumulates above the background level of fluorescence. The
.DELTA.Ct values are used as quantitative measurement of the
relative number of starting copies of a particular target sequence
in a nucleic acid sample when comparing the expression of RNA in a
cancer cell with that from a normal cell.
[0346] Results
[0347] The human Stra6 RNA (corresponding to DNA148380-2827) shows
strong over-expression in human colon tumor tissues, when compared
with corresponding normal human colon tissues. As shown in FIG. 11,
human Stra6 RNA (corresponding to DNA148380-2827) was found to be
over-expressed in all 14 human tumor colon tissues examined,
relative to RNA from matched normal colorectal mucosa from the same
patient. The over-expression varied between two- and 170-fold, and
in 7 out of 14 tumor tissue samples was at least about 10-fold. The
cycle threshold values obtained by quantitative PCR indicated that
Stra6 mRNA levels were extremely low or possibly absent in many of
the normal mucosa samples.
[0348] As a second method of detection, the products obtained after
completion of the quantitative PCR reactions (40 cycles each) were
subjected to electrophoresis in polyacrylamide gels and visualized
by ethidium bromide staining. As shown in FIG. 12A, using
expression of a housekeeping gene, glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) as the standard, substantially greater
amounts of PCR products were generated off Stra6 mRNA in tumor
samples compared to their normal counterparts. By contrast, a
comparable level of product from the internal control GAPDH mRNA
was generated from all samples.
[0349] Stra6 expression in colon adenocarcinomas was localized to
the epithelial tumor cells by in situ hybridization (ISH) performed
as follows. .sup.32P-labeled sense and antisense riboprobes were
transcribed from an 874 bp PCR product corresponding to nucleotides
432-1247 of the coding sequence of human Stra6 polypeptide encoded
by DNA148380-2827. Formalin-fixed, paraffin-embedded tissue
sections were processes as described previously (Pennica et al.,
Proc. Natl. Acad. Sci USA 95:14717-22 [1998]). The results are
shown in FIG. 12B.
[0350] As shown in FIG. 13, the human Stra6 RNA (corresponding to
DNA148380-2827) is also significantly over-expressed in various
breast, kidney, colon and lung tumor cell lines. "Relative RNA
Expression" means that the RNA expression in normal and tumor
tissues is shown relative to expression in an arbitrarily chosen
standard cell line SW480.
[0351] In situ hybridization results obtained in various tumor
sections are also shown in FIG. 16. Several tumor types other than
colon adenocarcinomas also showed high levels of Stra6 expression.
These included 3 of 3 melanomas (FIGS. 16A and B), 3 of 4
endometrial adenocarcinomas (FIGS. 16C and D), 2 of 3 ovarian
adenocarcinomas, and a Wilm's tumor of the kidney (FIGS. 16E and
F). The Stra6 in situ hybridization signal in these various tumors
was considerably greater than in colon adenocarcinomas consistent
with data showing relatively high expression levels in normal
kidney and uterus and low levels in normal colon. Since Stra6 was
detected in normal adrenal medulla, we also examined two
pheochromocytomas, which are tumors derived from this tissue. In
these tumors, Stra6 expression exceeded that of any other tumor or
tissue examined in this study (FIGS. 16G and H). Although Stra6 was
detected in normal kidney and was strongly expressed in Wilm's
tumor, it was not detected in renal cell carcinomas. In kidney
transitional cell carcinomas, tumor-associated stromal cells rather
than tumor epithelial cells expressed Stra6 (data not shown).
[0352] Because mRNA DNA148380-2827 encoding PRO10282 is
overexpressed in various tumors as well as in a number of tumor
derived cell lines, it is likely associated with tumor formation
and/or growth. As a result, antagonists (e.g., antibodies, organic
and inorganic small molecules, peptides and polypeptides, such as
Stra6 variants, antisense oligonucleotides) directed against the
protein encoded by DNA148380-2827 (PRO10282) or other naturally
occurring variants of this protein, such as PRO19578 encoded by
DNA148389-2827-1, are expected to be useful in diagnosis,
prevention and/or treatment of cancer particularly, without
limitation, colon, lung, breast and/or kidney cancer.
[0353] The efficacy of antagonists such as therapeutic antibodies
directed against the protein encoded by DNA148380-2827 (PRO10282)
could be enhanced by agents that stimulate the expression of the
gene encoding PRO10282. For example, treatment of human colorectal
cancer cell lines with 9-cis-retinoic acid or all-trans retinoic
acid resulted in a dramatic enhancement of the expression of the
Stra6 mRNA (FIG. 15). Thus, the treatment of cancer patients with
therapeutic antibodies directed against PRO10282 in combination
with the appropriate retinoids would be expected to enhance tumor
killing by the antibodies. The same will be true to antibodies
directed against other native Stra6 polypeptides over-expressed in
various tumors, such as the human splice variant encoded by
DNA148389-2827-1.
Example 13
Synergistic Induction of Stra6 by Wnt-1 and Retinoids
[0354] Materials and Methods
[0355] Cell Culture
[0356] C57MG and C57MG/Wnt-1 cells were grown in Dulbecco's
Modified Eagle medium supplemented with 10% fetal bovine serum, 2
mM L-glutamine, and 2.5 .mu.g/ml puromycin (Edge Biosystems). C57MG
cells with tetracycline-repressible Wnt-1 expression were grown in
complete medium without puromycin, supplemented with 400 .mu.ghml
G418 (Gibco BRL), 100 .mu.g/ml hygromycin B (Gibco BRL), and 50
ng/ml tetracycline (Korinek et al., Mol. Cell Biol. 18:1248-56
[1998]). For Wnt-1 induction studies, cells were washed with
phosphate buffered saline, cultured in tetracycline-free media for
10, 24, 48, 72, and 96 hours and then harvested. A 0-hour control
dish was maintained entirely in media containing tetracycline. All
dishes were simultaneously harvested and total RNA was extracted
for each time point. RT-PCR was carried out with Wnt-1, Stra6, and
GAPDH specific primers and probes on 100 ng total RNA from each
sample.
[0357] Human colon adenocarcinoma cell lines HCTI 16 and WiDr cells
were obtained from the American Type Culture Collection. HCT116
cells were maintained in McCoy's 5A media supplemented with 10%
fetal bovine serum (FBS). WiDr cells were maintained in Dulbecco's
minimal essential media (DMEM) supplemented with 10% FBS. For
retinoic acid induction studies, cells were plated at 10.sup.5
cells/60 mmu dish containing 2.5 ml of medium and allowed to grow
for 24 hours. Cells were treated with Vitamin D3, all-trans-RA
(Spectrum Laboratory Products), or 9-cis-RA (Toronto Research
Chemicals Inc.) (1 .mu.M final concentration in DMSO) for the
indicated times. Control cells were treated with an equal volume of
DMSO.
[0358] For the treatment of C57MG/Parent cells with Wnt-3a
conditioned media, cells were incubated in regular media or
conditioned media from L-cells or L-W3A cells in the presence or
absence of 1 .mu.M 9-cis-RA. Conditioned media was prepared as
previously described (Willert et al, Genes Dev. 13:1768-73 [1999]).
At 48 hours, cells were harvested by scraping into PBS containing
sodium fluoride and 25 vanadate and quick frozen in liquid
nitrogen. RNA was prepared using the RNAeasy kit (Qiagen),
including the additional DNaseI treatment on the columns according
to manufacturer's instructions.
[0359] Western Blotting
[0360] For the C57MG/Wnt-1 TET cell line, Wnt-1 expression was
induced by culturing cells in the absence of tetracycline for 0,
24, 48, or 72 hours. Cells were lysed in Triton X-100 lysis buffer
[20 mM tris-HCl (pH 8.0), 137 mM NaCl, 1% Triton X-100, 1 mM EGTA,
10% glycerol, 1.5 mM MgCl.sub.2, 1 mM dithiothreitol, 1 mM sodium
vanadate, 50 mM sodium fluoride, and complete protease inhibitor
cocktail (Boehringer Mannheim) and protein-equivalents were
subjected to SDS-PAGE and immunoblotting. Blots were incubated with
either 0.2 .mu.g/ml affinity purified rabbit polyclonal antibody
against .beta.-catenin (Rubinfeld et al., Science 262:1731-1734
[1993]), 0.1 82 g/ml anti-ERK2 monoclonal antibody (Transduction
Laboratories), or 1:2000 rabbit polyclonal antisera against
RAR.gamma.-1 (Affinity Bioreagents). For the WiDr cell line, cells
were treated with 1 .mu.M all-trans-RA for 48 hours and then lysed
in Triton X-100 lysis buffer and processed as indicated above.
Blots were incubated with 1:50 anti-Stra6 peptide B monoclonal
hybridoma culture supernatant (clone 12F4.2H9.1D5).
[0361] Immunohistochemistry
[0362] WiDr cells were treated with 9-cis-retinoic acid or DMSO,
then detached and pelleted by low-speed centrifugation. Cell
pellets were fixed overnight in 10% neutral buffered formalin,
dehydrated, and embedded in paraffin. Immunohistochemistry was
performed using anti-Stra6 peptide B monoclonal hybridoma culture
supernatant (clone 12F4.2H9.1D5) or nonspecific mouse isotype IgG2A
as primary antibodies, followed by detection using avidin-biotin
complex method with diaminobenzidine as chromogen (Vectastain Elite
Kit, Vector Laboratories) as described previously (Eberhard et al.,
Am. J. Pathol. 145:640-9 [1994]). Sections were counterstained with
hematoxylin.
[0363] Wnt-1 Transgenic Mice
[0364] Transgenic mice that express the Wnt-1 proto-oncogene in the
mammary gland typically exhibit hyperplastic lesions and develop
neoplasms in this tissue (Tsukamoto et al., Cell 55:619-625
[1988]). Such mice were used in the following experiments.
[0365] Results
[0366] Easwaran et al previously reported enhanced activation of a
synthetic retinoic acid responsive reporter gene when MCF-7 cells
were co-transfected with mutant .beta.-catenin and treated with
retinoids (Easwaran et al., Curr. Biol 9:1415-1418 [1999]).
Considering this, together with the original identification of
Stra6 as a retinoic acid inducible gene (Bouillet et al., Mech Dev.
63:173-186 [1997]), we asked whether retinoic acid could synergize
with Wnt-1 to increase the level of Stra6 in the C57MG cell line.
Treatment of parental C57MG cells with either 9-cis-RA or
all-trans-RA (ATRA) for 48 hours significantly increased the level
of Stra6 mRNA while DMSO and vitamin D3 had no effect (FIG. 17A).
As expected, the C57MG/Wnt-1 cells treated with either DMSO or
vitamin D3 exhibited enhanced levels of Stra6 mRNA relative to the
parent cell line. The level of Stra6 induction by Wnt-1 was
comparable to that observed on stimulation of the parental C57MG
cells with 9-cis-RA. However, 9-cis-RA treatment of the C57MG/Wnt-1
cell line induced a further 10-fold increase in Stra6 mRNA relative
to either untreated C57MG/Wnt-1 or 9-cis-RA treated C57MG parent
cells. Similar results were obtained with all-trans-RA.
[0367] It was possible that potential clonal variations in the
C57MG/Wnt-1 cell line, that were unrelated to Wnt-1 expression,
accounted for their differential response to retinoic acid relative
to parental control cells. To address this, we tested the response
of the parental C57MG cells to stimulation by soluble Wnt-3a in the
presence or absence of 9-cis-RA. Wnt-3a is a Wnt-1 homolog that
exhibits transforming properties similar to those of Wnt-1 and can
be produced as a soluble ligand in mouse L-cells. Conditioned media
from cultured L-cells expressing Wnt-3a, but not from control
L-cells, induced the expression of Stra6 in the C57MG cells (FIG.
17B). A slightly higher level of induction was observed on
treatment of C57MG cells with 9-cis-RA. However, the combination of
9-cis-RA and Wnt-3a resulted in levels of Stra6 transcript vastly
exceeding that seen with either agent alone.
[0368] If the induction of Stra6 in the retinoic acid treated C57MG
/Wnt-1 cells was potentiated by increased .beta.-catenin levels,
then one might expect a similar induction of Stra6 in response to
retinoic acid in human colon carcinoma cells containing mutations
in either .beta.-catenin or APC. To determine whether this occurs,
Stra6 mRNA levels were measured before and after retinoic acid
treatment in HCT116 cells, which carry an activating mutation in
.beta.-catenin, and in WiDr cells, which have lost both copies of
wild-type APC. In both cell lines, a significant increase in Stra6
mRNA levels was seen following treatment with either ATRA or
9-cis-RA compared to DMSO or vitamin D3 (FIG. 17C). The activation
of the Stra6 gene by ATRA in the HCT116 cell line was confirmed by
in situ hybridization (FIG. 17D). Induction of the predicted 73 kDa
Stra6 protein band in WiDr cells treated with ATRA was detected by
Western blot analysis with a Stra6 specific monoclonal antibody
(FIG. 17E). Immunohistochemical analysis of the WiDr cells revealed
that the increase in Stra6 protein in response to retinoic acid was
localized to the plasma membrane (FIG. 17F).
[0369] The RAR.gamma. gene has been proposed as a target for Wnt
signaling in Xenopus embryos, and the induction of Stra6 by
retinoids was shown to be dependent upon the presence of this
retinoic acid receptor subtype (McGrew et al., Mech. Dev. 87:21-32
[1999]; Taneja et al., Proc. Natl. Acad. Sci. USA 92:7854-8
[1995]). Together, these observations suggest that the synergistic
activation of Stra6 by Wnt and retinoids might be due to the
activation of RAR.gamma. expression by Wnt signaling. To determine
whether Wnt-1 signaling had any influence over the levels of
RAR.gamma. in mammalian cells, we performed Western blots for the
receptor on lysates prepared from C57MG cells that conditionally
express Wnt-1. Upon Wnt-1 expression, a protein reactive with
antibody specific to RAR.gamma.-1, and migrating with an apparent
molecular mass of 64 kDa, was induced at 24 hours and its
expression was increased at 48 hours (FIG. 18A). We also analyzed
hyperplastic mammary glands and mammary gland tumors obtained from
Wnt-1 transgenic mice and detected elevated levels of RAR.gamma.
mRNA in these tissues relative to normal mammary gland (FIG. 18B).
The level of RAR.gamma. transcript present in equivalent amounts of
RNA isolated from 19 adenocarcinomas was assessed by quantitative
PCR. Notably, RAR.gamma. mRNA expression was increased
approximately 2-4 fold in 14 of the 19 (74%) of human colon tumors
examined compared to normal human colon tissue (FIG. 18C). These
results demonstrate that Wnt-1 signaling promotes the expression of
RAR.gamma. that the receptors are elevated in mouse and human
tumors that are driven by the Wnt-1 pathway.
[0370] Discussion of Experimental Findings
[0371] Gene expression profiling approaches are based on unbiased
detection of mRNA transcripts and can therefore lead to unexpected
insights into the mechanisms by which gene activation occurs. Here
it has been shown that Wnt-1 promoted the induction of the retinoic
acid responsive gene Stra6, suggesting a connection between
signaling pathways elicited by Wnt and retinoic acid. This
connection was further supported by demonstrating a synergistic
induction of Stra6 by a combination of Wnt and retinoic acid. There
are at least three alternative explanations that could account for
this synergy: i) transcription factors directly responsive to Wnt
such as the TCF/LEFs might bind to and activate promoter elements
in the Stra6 gene; ii) signaling components in the Wnt pathway
might directly interact with the appropriate retinoic acid receptor
(RAR) and potentiate gene activation mediated by the RAR; or iii)
signaling by Wnt-1 could activate expression of the appropriate
RAR, which could then sensitize cells to retinoic acid. Although
this final proposal is favored because the data presented herein
show that Wnt-1 signaling rapidly induces expression of the
RAR.gamma. receptor, the present invention is not limited by any
particular theory or mechanism of action.
[0372] Previous work has shown that specific disruption of the
RAR.gamma. gene greatly reduced induction of the Stra6 gene by
retinoids, which was then rescued on re-expression of this receptor
(Taneja et al, supra). However, it remains possible that mechanisms
in addition to the Wnt-1-induced expression of RAR.gamma. may also
contribute to the observed synergy. The proposal that
.beta.-catenin binds to retinoic acid receptors is attractive, but
we have not observed any specific association of endogenous
RAR.gamma. with .beta.-catenin in our cell lines. However, the
binding of .beta.-catenin to RAR.gamma., as was shown in vitro
(Easwaran et al, 1999, supra), might relate to the activation of
Stra6 by Wnt-1. The expression pattern of Stra6 in transgenic
animals null for RAR.gamma. was more widespread than that observed
in wild-type littermates, and induction of Stra6 by retinoic acid
was enhanced in cells from the null animals compared to controls
(Bouillet et al., 1997, supra; Taneja et al., 1995, supra). Thus
RAR.gamma. might be inhibitory to Stra6 expression, and activation
of .beta.-catenin by Wnt-1 could potentially relieve this
inhibition if .beta.-catenin inhibited RAR.gamma. function.
[0373] The synergistic induction of a retinoic acid responsive gene
by Wnt-1 signaling implies that human cancers that harbor genetic
defects in the Wnt-1 pathway would exhibit overexpression of Stra6.
The vast majority of colorectal tumors contain mutations in the
genes coding for either the APC tumor suppressor or .beta.-catenin
(Polakis, Curr. Opin. Genet. Dev. 9:15-21 [1999]) and, accordingly,
we have detected overexpression of the Stra6 transcript in 14 of 14
colorectal tumors relative to matched normal tissue (see Example
12). Activating mutations in .beta.-catenin have also been
identified in cancers of the ovary and endometrium, Wilm's kidney
tumors and melanomas, demonstrating that defects in Wnt-1 signaling
contribute to the progression of these cancers (Kobayashi et
al.Jpn. J. Cancer Res. 90:55-9 [1999]; Koesters et al., Cancer Res.
59:3880-2 [1999]; Palacios and Hamallo, Cancer Res. 58:1344-[1998];
Rimm et al. Am. J. Pathol. 154:325-9 [1999]; Rubinfeld et al.
Science 262:1731-1734 [1993]; Wright et al. Int. J. Cancer 82:625-9
[1999]). All four of these human cancers displayed overexpression
of Stra6 mRNA as determined by in situ hybridization (see Example
12). Pheochromocytoma, a tumor derived from the adrenal medulla,
exhibited extremely high levels of Stra6 mRNA, however, the status
of these tumors with respect to mutations in the Wnt-1 signaling
pathway has not been reported. It is intriguing that the cell types
that give rise to melanomas and pheochromocytomas share in common
an embryological derivation from the neural crest. Notably, Wilms'
tumor of the kidney, pheochromocytoma, and endometrial carcinomas
all arise in organs in which Stra6 is normally expressed. This
suggests that tumorigenic signaling, such as that driven by the
Wnt-1 pathway, might interact with signals responsible for
differentiation, thereby hyper-activating the expression of
Stra6.
[0374] The human Stra6 protein resides at the cell surface, as
determined by the staining of colorectal cancer cells with
monoclonal antibodies specific to human Stra6 protein. Moreover,
the staining intensity observed at the cell membrane was increased
after treatment of cells with retinoic acid. Thus, Stra6 likely
encodes a multi-pass transmembrane protein that is localized to the
cell surface.
[0375] It is noted that ISRL, one of the genes identified in the
present screen, has been found to be contiguous with the Stra6
gene. It is believed that any other gene flanking Stra6 and/or
ISRL, or any gene located in close proximity to Stra6 and/or ISRL
is a good candidate for the methods of the present invention.
Example 14
Identification of Further Genes Synergistically Induced by Wnt-1
and Retinoic Acid Receptor (RAR) Signaling
[0376] The data disclosed in Example 13 above show that endogenous
retinoic acid responsive gene Stra6, was activated upon Wnt-1
expression. Moreover, stra6 was synergistically activated by a
combination of retinoic acid and Wnt-1. The synergistic activation
of stra6 did not require the overexpression of intracellular
signaling components and is therefore mediated entirely by
endogenous signaling molecules responsive to their corresponding
receptors. Also, human colorectal cancer cell lines that exhibit
hyperactive Wnt signaling responded to retinoic acid treatment by
producing high levels of Stra6 protein. This suggests that genuine
cross talk might occur between the RAR and Wnt signaling pathways
under normal physiological or pathological conditions.
[0377] That the induction of Stra6 expression by retinoic acid was
more robust on an oncogenic background prompted us to consider
potential therapeutic applications where this effect could be
exploited. As the Stra6 gene codes for a cell surface protein, an
appropriate application is immunotherapy in cancer. The proven
utility of therapeutic antibodies in treatment of human cancer in
the clinic has recently spurned intense activity aimed at the
development and refinement of immunotherapeutics. Ideally, these
therapies require the presence of cell surface antigens expressed
on the cancer cells at significantly higher levels than that
present on normal tissues throughout the body. Such criteria for
differential expression on tumors relative to normal tissue will
obviously limit the number of antigens considered desirable as
targets for immunotherapy. Therefore, reagents that selectively
enhance the level of antigen expression of cancer cells relative to
normal cells are expected to improve the therapeutic index for
immunotherapeutics directed against these antigens. To this end we
performed a screen to identify further antigens that are
preferentially upregulated by the treatment of Wnt-1 transformed
cells with retinoic acid.
[0378] Materials and Methods
[0379] Cell Culture
[0380] A murine C57MG breast epithelial cell line was engineered to
express the Wnt-1 proto-oncogene upon withdrawal of tetracycline
from the cell culture media (Korinek et al., Mol Cell Biol
18:1248-56 (1998)). The cells were grown in 10 cm dishes in
Dulbecco's Modified Eagle medium supplemented with 10% fetal bovine
serum, 2 mM L-glutamine, 100 units/ml penicillin/streptomycin, 200
ng/ml tetracycline, 400 .mu.g/ml G418, and 100 .mu.g/ml hygromycin
B until approximately 60% confluent. Cells were washed with
phosphate buffered saline, cultured in tetracycline-free media for
48 hours either in the presence of 1 .mu.M all-trans-retinoic acid
(ATRA) or an equal volume of DMSO and then harvested. Control cells
were maintained entirely in media containing tetracycline either in
the presence of 1 .mu.M ATRA or DMSO. All dishes were
simultaneously harvested and total RNA was extracted. The growth
and treatment of cells and the purification of RNA from them was
performed 2 independent times. In addition to these experiments, a
time course was performed in which the cells grown under the
conditions described above were harvested at 24, 48 and 72 hours.
The results from the 48 hour time point from this experiment alone
with those from the two 48 hour experiments described above
comprise the triplicate data set that is presented in the results
section.
[0381] Total RNA Extraction
[0382] Cells were lysed in 3.5 ml of lysis buffer (4M guanidine
thiocyanate, 25 mM sodium citrate, 0.5% N-lauryl sarcosine, 0.7%
2-mercaptoethanol) and layered on 1.5 ml of 5.7 M cesium
chloride/50 mM EDTA (pH8.0) solution. The lysate was spun at
150,000.times.g overnight. The RNA pellet was dried, resuspended in
water, phenol:chloroform extracted, and ethanol precipitated. The
RNA was resuspended in water to a final concentration of 1 mg/ml
and stored at -70.degree. C.
[0383] Oligonucleotide Array
[0384] Approximately 10 mg of each RNA sample served as starting
material for the preparation of probes required for oligonucleotide
array analysis on the miroarray system of Affymetrix (Santa Clara,
Calif.). Probes were prepared according to previously described
protocols (Wodicka et al. Nat Biotechnol 15:1359-1367 (1997)).
Following hybridization, the arrays were washed and stained with
streptavidin-phycoerythrin and then scanned with the Gene Array
scanner (Agilent Technologies). Default parameters provided in the
Affymetrix data analysis software package were applied in
determining the signal intensities, referred to as average
differences, and the fold differences for the 12,000 gene probe
sets represented on the Affymetrix "A" Mu74 chip. The average
differences obtained with probes derived from cells expressing
Wnt-1 in the absence or presence or ATRA, or treated with ATRA in
the absence of Wnt-1 expression, were base-lined against average
differences obtained from untreated control cells to generate the
fold difference value for each gene call.
[0385] Reverse Transcriptase-PCR (RT-PCR) Analysis
[0386] Confirmation of gene expression was performed using
quantitative RT-PCR using Taqman assay reagents in an ABI 7700
Sequence Detector from Perkin-Elmer, Applied Biosystems. RT-PCR
reactions consisted of 100 ng RNA, 5 .mu.l 10.times. Taqman Buffer
A, 300 .mu.M of each dNTP, 5 mM MgCl.sub.2, 10 units of RNase
inhibitor, 12.5 units of MuLV Reverse Transcriptase, 1.25 units of
AmpliTaq Gold DNA Polymerase, 200 nM probe, and 500 nM primers.
Reaction conditions consisted of reverse transcription at
48.degree. C. for 30 minutes, denaturation at 95.degree. C. for 10
minutes, and 40 cycles of 95.degree. C. for 25 seconds and
65.degree. C. for 1 minute. Reaction products were analyzed on
4-20% polyacrylamide gels.
[0387] Fold induction was obtained by using the
.DELTA..DELTA.C.sub.t method in which all samples are first
normalized to the level of glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) in each sample. Relative normalized units were then
compared between the experimental sample and its control. Mouse
GAPDH, Ephrin B1, 4-1BB ligand, and ISLR primer and probe sequences
are as follows:
4 GAPDH forward primer: 5'-TGCACCACCAACTGCTTAG-3' (SEQ ID NO: 17);
reverse primer: 5'-GGATGCAGGGATGATGTTC-3' (SEQ ID NO: 18); probe:
5'-CAGAAGACTGTGGATGGCCCCTC-3' (SEQ ID NO: 19). Ephrin B1 forward
primer: 5'-GTAGGCCAGGGCTATtTCTG-3' (SEQ D NO: 20); reverse primer:
5'-TGTCTCATGAGGGTCCAAAA-3- ' (SEQ ID NO: 21); probe:
5'-TGGCGCTCTTCTCTCCTCGGT-3' (SEQ ID NO: 22). 41BB ligand forward
primer: 5'-TCGCCAAGCTACTGGGTAA-3' (SEQ ID NO: 23); reverse primer:
5'-CTTGGCTGTGCCAGTTCA-3' (SEQ ID NO: 24); probe:
5'-AACCAAGCATCGTTGTGCAATACAACTC-3' (SEQ ID NO: 25). ISLR forward
primer: 5'-GCCAGTACAGGATCTGGAAAG-3' (SEQ ID NO: 26); reverse
primer: 5'-CATATCTCATCAGAGAGCATCTAAAA-3' (SEQ ID NO: 27); probe:
5'-AAG CTT TTA GCC TGC CCA GCC A-3' (SEQ ID NO: 28).
[0388] Western Blotting
[0389] Following indicated treatment, cells were lysed in Triton
X-100 lysis buffer (20 mM tris-HCl (pH 8.0), 137 mM NaCl, 1 %
Triton X-100, 1 mM EGTA, 10% glycerol, 1.5 mM MgCl.sub.2, 1 mM
dithiothreitol, 1 mM sodium vanadate, 50 mM sodium fluoride, and
Complete protease inhibitor cocktail (Boehringer Mannheim, Mannheim
Germany)) and protein-equivalents were subjected to SDS-PAGE and
immunoblotting. Blots were incubated with 0.1 .mu.g/ml anti-ERK2
monoclonal antibody (Transduction Laboratories, Lexington, Ky.),
anti-myc tag monoclonal antibody. Blots were developed using the
ECL system (Amersham).
[0390] Luciferase Assay
[0391] To determine RAR-dependent transactivation, Cos-7 cells were
cotransfected with the indicated expression plasmids and the TREpal
luciferase reporter construct (Beyers) using Effectene (Qiagen)
transfection reagent per the manufacturer's instructions.
Expression plasmids have been described elsewhere. Cells were
treated with 1 .mu.M ATRA or DMSO control on the day of
transfection and harvested 72 hours later by lysis in Triton X-100
lysis buffer. Luciferase activity in 10 .mu.l of lysate was
analyzed using a Tropix TR717 microplate luminometer. Activity was
normalized to Renilla luciferase activity produced by
cotransfection with SV40-Renilla luciferase. Activation of
LEF/TCF-dependent transcription was determined by cotransfection
with indicated plasmids and Top-tk-luciferase plasmid. Activity was
determined as described above.
[0392] Results
[0393] The murine C57MG breast epithelial cell line undergoes
morphological transformation in response to the expression of
various Wnt genes (Wong et al., Mol Cell Biol 14:6278-6286 (1994)).
A version of this cell line that was engineered to conditionally
express Wnt-1 in response to the removal of tetracycline from the
culture medium was employed for the gene expression profiling
experiments. To identify genes that were preferentially activated
by the combination of retinoic acid and Wnt-1 signaling, four
different conditions were established for the treatment of cells.
As a control, cells were left in medium containing tetracycline
plus DMSO, the vehicle for retinoic acid. A second dish of cells
was treated with 100 nM all-trans-retinoic acid (ATRA) in the
presence of tetracycline, while a third dish of cells received DMSO
control and the tetracycline was removed to activate expression of
Wnt-1. Finally, a fourth dish of cells was treated with ATRA and
the tetracycline was removed. Following a forty eight hour
incubation period, cells were harvested and RNA was extracted and
purified. Probes synthesized from the RNA was hybridized to the
Affymetrix Mouse Gene Chip "A" Mu74 containing 12,000
oligonucleotide probe sets. The experiment, as initiated from the
growth and treatment of cells, was performed three independent
times. Data is presented for mRNA transcripts that underwent at
least a two-fold increase or a 50% decrease relative to the
untreated cells in all three experiments. In addition, a time
course was performed in which RNA was collected from treated cells
at 24, 48 and 72 hours. These data are included only for select
genes.
[0394] Treatment of the C57MG cells with retinoic acid alone
resulted in the robust activation of numerous genes (Table 1).
5TABLE 1 Gene Acc. # Fold Change Std. Dev Up regulated by retinoic
acid thioether S-methyltransferase M88694 154.57 204.10 Casein
kappa M10114 33.67 35.64 3naiotensinoaen AF046887 27.63 17.34
retinal short-chain dehydrogenase/reductase X95281 25.67 28.78
Ribonuclease I X60103 18.60 10.87 FGF-1 M30641 18.43 16.46
LPS-binding protein X99347 15.10 6.74 cytochrome P450. 2f2 M77497
10.77 8.30 Death-asociated Drotein kinase 2 AB018002 10.67 10.87
Decorin X53929 8.07 4.46 11beta-hydroxysteroid
dehydropenase/carbonyl reductase X83202 7.97 4.02 autotaxin
AW122933 7.90 5.72 type 2 deiodinase AF096875 7.63 5.71 v-erb-a
homolog-like 3 X74134 7.00 2.65 aldehvde dehydrogenase 3 AF033034
6.43 3.07 3-O-sulfotransferase AF019385 6.13 3.97 ficolin-A
AB007813 5.87 3.69 lysosomal acid lipase Z31689 5.60 2.46
osteoglycin D31951 4.43 2.15 ATP-binding cassette 1, sub-family A.
member 1 (Abca1) gene A1845514 4.40 2.12 kallikrein V00829 4.33
1.22 Catheosin B A1861255 3.90 1.21 Complement component C3 K02782
3.83 2.67 Procollogen, type IV, alpha 5 Z35168 3.53 1.01
diacylglycerol acyltransferase AF078752 3.40 1.46 chaperonin
containing TCP-1 epsilon subunit AW049373 3.33 1.88 VEGF C U73620
3.10 0.95 Glutathione-S-transferase, alpha 1 L06047 3.10 0.80
ceruloplasmin U49430 3.03 1.30 sorting nexin 10 A1746846 2.90 1.15
receptor interacting protein 140 AF053062 2.83 1.62 megakaryocyte
potentiating factor D86370 2.73 1.35 annexin VI X13460 2.67 1.50
MG87 AW124268 2.63 0.75 prefoldin AB023957 2.60 0.44 myristoylated
alanine rich PKC substrate M60474 2.60 0.78 Rho guanine nucleotide
exchange factor AJ010045 2.43 0.55 solute carrier family 12, member
2 U13174 2.40 0.61 quanylate nucleotide binding protein 3 (Gbp3)
AW047476 2.37 0.58 isovaleryl CoA dehydrogenase (lvd) AW047743 2.37
1.16 alpha-2,3-sialyltransferase 028941 2.37 0.35 Retinoblastoma-I
M26391 2.37 0.40 Caspase 3 U54803 2.37 1.07 Acyl-CoA synthetase
AA619207 2.30 0.36 Lysosomal membrane glycoprotein 2 M32017 2.30
0.56 TM6P1 AA881018 2.23 0.47 Mad4 U32395 2.17 0.21 interleukin
1-beta converting enzyme L28095 2.00 0.26 Up regulated by wnt
autotaxin-t AW122933 4.27 1.52 4-1bb Ligand L15435 3.53 1.21
prefoldin (MM-1) AB023957 3.00 0.26 Semaphorin E X85994 2.90 0.90
BTEB2 (IKLF) AA611766 2.80 0.26 G28K RHO-RAS family GTP-binding
protein AW121294 2.73 0.61 SSG-1 steroid sensetive gene-1 protein
AW122012 2.57 1.10 TCF-1 A1019193 2.57 0.81 GADD45 gamma AF055638
2.43 0.25 Frizzled-2 AW123618 2.23 0.23 ets-2 J04103 2.20 0.40
TSA-1 U47737 2.13 1.01 adenosine kinase AW121801 1.87 0.35 Down
regulated by wnt Contactin I X14943 -7.17 4.65 Protacilandin E
receptor, EP4 Dl 3458 -5.40 2.69 Orosomucoid 1 M27008 -3.93 2.51
calpain-like protease Y12582 -3.83 1.72 RGS2 U67187 -3.30 1.30
Small inducible cytokine A7 X70058 -3.13 1.06 Lymphocyte antigen 84
D13695 -2.97 1.68 complement factor H-related protein M29010 -2.50
0.79 Small inducible cytokine B subfamily, member 5 U27267 -2.50
0.52 Complement component factor h M12660 -2.47 1.61 Ceruloplasmin
U49430 -2.30 0.53 embigin AW061330 -2.13 0.31 Prostaglandin F
receptor D17433 -2.03 0.49
[0395] Some of these genes, such as decorin (Pearson, D. and Sasse,
J., J Biol Chem 267:25364-25370 (1992)), COUPTF-1 (Clotman et al.,
Neurotoxicol Teratol 20:591-599 (1998)), 11-beta-hydroxysteroid
dehydrogenase (Tremblay et al., Biol Reprod 60:541-545 (1999)),
3-O-sulfotransferase (Zhang et al., J Biol Chem 273:27998-28003
(1998)), Abcal (Wade, D. P. and Owen, J. S., Lancet 357:161-163
(2001)) and ceruloplasmin (Koj et al., Biol Chem Hoppe Seyler
374:193-201 (1993)) have been reported previously to be upregulated
by retinoic acid in other cell types. The identification of these
genes in our screen demonstrates that the C57MG cells respond
appropriately to retinoic acid and suggests that common endpoints
for retinoic acid signaling exist in diverse cell types. Additional
genes induced by retinoic acid, such as retinal short-chain
dehydrogenase and aldehyde dehydrogenase 3, code for enzymes that
are involved in the metabolism of retinoids and might, therefore,
represent feedback or feed-forward responses to retinoic acid
itself (Duester, Eur J Biochem 267:4315-4324 (2000)). Finally,
several thergenes that were induced by retinoic acid, such as
autotaxin, thioether S-methyl transferase and angiotensiogen, bear
no obvious connection to retinoic acid signaling or metabolism and
have not been previously reported to be activated by receptors
responsive to retinoic acid.
[0396] To identify genes induced by Wnt-1, tetracyline was removed
from the cells in the absence or retinoic acid. Under these
conditions, 13 transcripts were identified that were expressed at
2-fold or higher levels relative to control cells in all three
experiments. This is likely an underestimate of the number of genes
that are actually induced by Wnt-1 signaling in these cells.
Indeed, numerous additional genes, including cyclin D1, which is a
target of Wnt-1 signaling (Tetsu, O. and McCormick, F., Nature
398:422-426 (1999)), were identified in only 2 of the 3 experiments
or fell short of the 2-fold cutoff. Two of the genes identified in
our screen, the TCF1 and BTEB2 transcription factors, were
previously reported to be targets of Wnt signaling (Ziemer et al.,
Mol Cell Biol 21:562-574 (2001); Roose et al., Science
285:1923-1926 (1999)).
[0397] Splice variants of TCF1 that bind DNA but lack
.beta.-catenin binding sites have provide negative feedback in Wnt
signaling and thus function as tumor suppressors. In addition, one
of the Wnt receptors, frizzled-2, was also upregulated by Wnt
signaling in the C57MG cell line. Some of the genes upregulated by
Wnt-1, such as GADD45 gamma and 4-1BB ligand, might represent a
response to inappropriate growth signaling due to the
overexpression of Wnt-1. The 4-1BB ligand (CD137) is a TNF
superfamily member that is expressed on various human carcinoma
cell lines and stimulates T-cell responses in tumor rejection. The
activation of GADD45.gamma./CR6, suggests that Wnt signaling
promotes errors affecting the fidelity of DNA replication. Indeed,
p53 mutations cooperate with Wnt-1 overexpression to produce tumors
in mice that exhibit chromosomal instability (Donehower et al.,
Genes Dev 9:882-895 (1995)). Two additional genes upregulated by
Wnt-1, semaphorin E and autotaxin, have been implicated in human
tumor progression as factors contributing to the motility and
metastatic spread of cancer cells (Martin-Satue, M. and Blanco, J.,
J Surg Oncol 72:18-23 (1999); Yamada et al., Proc Natl Acad Sci USA
94: 14713-14718 (1997)); Nam et al., Oncogene 19:241-247
(2000)).
[0398] As described above, we identified stra6 as a gene activated
by Wnt-1 signaling. Stra6 was originally identified in a screen
designed to detect mRNA transcripts induced by retinoic acid
receptor signaling (Bouillet et al., Dev Biol 170:420-433 (1995)).
Although we found that retinoic acid induced the expression of
stra6, we also demonstrated its synergistic activation by a
combination of Wnt-1 and retinoic acid (see Example 13 above).
Therefore, we were interested in whether genes in addition to stra6
could be activated in a like fashion. Consistent with our previous
findings, stra6 was activated by either retinoic acid or Wnt-1,
while the combination of these agents resulted in expression levels
greatly exceeding that observed with either agent alone (FIG. 19).
A similar pattern of expression was observed for autotaxin, which
underwent modest activation in the presence of retinoic acid or
Wnt-1 alone, but more robust induction in response to both stimuli
(FIG. 20). The synergistic activation of autotaxin was confirmed by
performing RT-PCR analysis on RNA extracted from C57MG cells
subjected to same treatment as those utilized in the expression
profiling screen. The 41BB ligand and ephrin b1 were also
synergistically induced by Wnt-1 and retinoic acid, although
retinoic acid alone appeared not to have a significant affect on
their expression (FIGS. 21 and 22). The synergistic activation of
these two genes was also confirmed by RT-PCR. In the case of ephrin
b1, antibody was commercially available, and was used to
demonstrate synergistic activation at the level of protein
expression (FIG. 22). In contrast to the activation of ephrin b1
and 41BB ligand, the ISLR gene was responsive to retinoic acid, but
not to Wnt, while the combination of both agents resulted in
activation that exceeded that of retinoic acid alone (FIG. 23).
Some genes, such as M-ras and the tight junction protein ZO-1,
appeared not to respond significantly to either retinoic acid or
Wnt-1 alone, but were only activated by combined treatment (FIG.
24).
[0399] It is apparent that certain genes undergo synergistic
activation by a combination of retinoic acid and Wnt-1, although
the signaling mechanism that mediates this effect has not been
clearly defined. We have found increased levels of RARg protein in
cells activated by Wnt-1 expression (data not dislosed). Therefore,
cells activated by Wnt might be more sensitive to retinoic acid due
to the higher levels of RARg. However, the synergistic activation
of stra6 by Wnt-1 and retinoic acid preceded any observable
increase in RARg suggesting that additional mechanisms were at
work. Moreover, the ability of Wnt-1 alone to activate stra6 was
inhibited by a pan-antagonist of RAR signaling. This suggests that
even in the absence of exogenously added RA, Wnt-1 was dependent
upon retinoic RAR signaling for the induction of stra6. Therefore,
it is conceivable that Wnt facilitates RAR signaling in a manner
that does not involve the canonical Wnt-1 pathway in which
.beta.-catenin interacts the TCF/LEF transcription factors. To
examine this we overexpressed an oncogenic mutant of .beta.-catenin
and measured the activation of an RAR response element (RARE) in
the presence and absence of overexpressed LEF. Expression of
.beta.-catenin activated the RARE as determined by the increased
production of luciferase (FIG. 25A). However, coexpression of
.beta.-catenin did not potentiate RARE activity but, conversely,
inhibited its activation by .beta.-catenin. This was not the case
for a LEF responsive element TopFlash, which was activated further
upon coexpression of LEF with b-catenin (FIG. 25B). The results
indicate that .beta.-catenin is involved in the activation of some
retinoic acid responsive genes in a manner that does not involve
LEF. The ability of LEF to inhibit the activation of RARE by
.beta.-catenin is most likely be due to competitive binding as a
b-catenin binding site on LEF was required for this effect (data
not shown). This result suggests that LEF binds to b-catenin in a
manner that precludes its interaction with RAR signaling components
and thereby prevents it from potentiating RAR signaling.
Example 15
Induction of Stra6 in Explanted Mammary Gland Tumors
[0400] The results with the C57MG cell line presented in Example 14
above, suggest that tumors driven by Wnt signaling are likely to
respond to retinoic acid by expressing high levels of mRNA
transcripts that are synergistically induced by Wnt and retinoic
acid. To examine this, mammary tumors derived from Wnt-1 transgenic
mice prepared as described in Example 13, were transplanted to the
mammary fad pad of naive mice that were subsequently administered
retinoic acid. Administration of retinoic acid (ATRA) but not
vehicle control resulted in the upregulation Stra6 mRNA in the
transplanted mouse mammary tumors but did not have a significant
effect on Stra6 expression in normal mammary tissue.
[0401] The data presented in FIG. 26 shows the effect of induction
of Stra6 following peritumor injection of 100 mg/kg and 400 mg/kg
(about 10 mg total/25 gram mouse) all-trans retinoic acid. We have
also found that a control gene, retinal DHR, which is responsive to
retinoic acid alone, was induced in normal mammary tissue (data not
shown). In FIG. 26 "Tum" stands for transplanted mouse mammary
tumor; "MG" stands for normal mammary tissue, "PT" stands for
peri-tumor, and the numbers immediately preceding the "MG" and
"Turn" are used to designate the particular animals from which
samples were taken. The data presented are mRNA expression levels
normalized to the housekeeping gene, GAPDH. In particular, standard
curves were used to determine relative levels of expression for
each gene of interest as well as the GAPDH housekeeping gene for
each sample analyzed. Relative normalized units were obtained by
dividing the mRNA level for the gene of interest by the GAPDH mRNA
level. Tumors and normal adjacent mammary glands were harvested 8
hours after treatment.
[0402] FIG. 27 shows that administration of ATRA to nude mice
bearing WiDr xenografts also induces Stra6. Specifically the mice
were given ATRA per orum (PO) at 400 mg/kg. The tumors were
harvested 12 hours following treatment. In this experiment oral
administration of ATRA (PO) was tested as well as peritumoral
administration (data not shown). The data presented in FIG. 27 show
that oral administration of 400 mg/kg of ATRA selectively
upregulates Stra6 mRNA in WiDr xenografts as compared to untreated
control. mRNA expression levels were normalized as discussed
above.
[0403] Deposit of Material
[0404] The following materials have been deposited with the
American Type Culture Collection, 10801 University Blvd., Manassas,
Va. 20110-2209, USA (ATCC):
6 Material ATCC Dep. No. Deposit Date DNA148380-2827 PTA-1181
January 11, 2000 DNA148389-2827-1 PTA-1402 February 23, 2000
[0405] These deposits were made under the provisions of the
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for the Purpose of Patent Procedure and the
Regulations thereunder (Budapest Treaty). This assures maintenance
of a viable culture of the deposit for 30 years from the date of
deposit. The deposit will be made available by ATCC under the terms
of the Budapest Treaty, and subject to an agreement between
Genentech, Inc. and ATCC, which assures permanent and unrestricted
availability of the progeny of the culture of the deposit to the
public upon issuance of the pertinent U.S. patent or upon laying
open to the public of any U.S. or foreign patent application,
whichever comes first, and assures availability of the progeny to
one determined by the U.S. Commissioner of Patents and Trademarks
to be entitled thereto according to 35 U.S.C. .sctn.122 and the
Commissioner's rules pursuant thereto (including 37 C.F.R.
.sctn.1.14 with particular reference to 886 OG 638).
[0406] The assignee of the present application has agreed that if a
culture of the materials on deposit should die or be lost or
destroyed when cultivated under suitable conditions, the materials
will be promptly replaced on notification with another of the same.
Availability of the deposited material is not to be construed as a
license to practice the invention in contravention of the rights
granted under the authority of any government in accordance with
its patent laws.
[0407] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. The present invention is not to be limited in scope by
the construct deposited, since the deposited embodiment is intended
as a single illustration of certain aspects of the invention and
any constructs that are functionally equivalent are within the
scope of this invention. The deposit of material herein does not
constitute an admission that the written description herein
contained is inadequate to enable the practice of any aspect of the
invention, including the best mode thereof, nor is it to be
construed as limiting the scope of the claims to the specific
illustrations that it represents.
[0408] It is understood that the application of the teachings of
the present invention to a specific problem or situation will be
within the capabilities of one having ordinary skill in the
pertinent art in light of the teachings contained herein. The
examples of the methods and products of the present invention
should not be construed to limit the invention.
Sequence CWU 1
1
28 1 2732 DNA Homo sapiens 1 agtcccagac gggcttttcc cagagagcta
aaagagaagg gccagagaat gtcgtcccag 60 ccagcaggga accagacctc
ccccggggcc acagaggact actcctatgg cagctggtac 120 atcgatgagc
cccagggggg cgaggagctc cagccagagg gggaagtgcc ctcctgccac 180
accagcatac cacccggcct gtaccacgcc tgcctggcct cgctgtcaat ccttgtgctg
240 ctgctcctgg ccatgctggt gaggcgccgc cagctctggc ctgactgtgt
gcgtggcagg 300 cccggcctgc ccagccctgt ggatttcttg gctggggaca
ggccccgggc agtgcctgct 360 gctgttttca tggtcctcct gagctccctg
tgtttgctgc tccccgacga ggacgcattg 420 cccttcctga ctctcgcctc
agcacccagc caagatggga aaactgaggc tccaagaggg 480 gcctggaaga
tactgggact gttctattat gctgccctct actaccctct ggctgcctgt 540
gccacggctg gccacacagc tgcacacctg ctcggcagca cgctgtcctg ggcccacctt
600 ggggtccagg tctggcagag ggcagagtgt ccccaggtgc ccaagatcta
caagtactac 660 tccctgctgg cctccctgcc tctcctgctg ggcctcggat
tcctgagcct ttggtaccct 720 gtgcagctgg tgagaagctt cagccgtagg
acaggagcag gctccaaggg gctgcagagc 780 agctactctg aggaatatct
gaggaacctc ctttgcagga agaagctggg aagcagctac 840 cacacctcca
agcatggctt cctgtcctgg gcccgcgtct gcttgagaca ctgcatctac 900
actccacagc caggattcca tctcccgctg aagctggtgc tttcagctac actgacaggg
960 acggccattt accaggtggc cctgctgctg ctggtgggcg tggtacccac
tatccagaag 1020 gtgagggcag gggtcaccac ggatgtctcc tacctgctgg
ccggctttgg aatcgtgctc 1080 tccgaggaca agcaggaggt ggtggagctg
gtgaagcacc atctgtgggc tctggaagtg 1140 tgctacatct cagccttggt
cttgtcctgc ttactcacct tcctggtcct gatgcgctca 1200 ctggtgacac
acaggaccaa ccttcgagct ctgcaccgag gagctgccct ggacttgagt 1260
cccttgcatc ggagtcccca tccctcccgc caagccatat tctgttggat gagcttcagt
1320 gcctaccaga cagcctttat ctgccttggg ctcctggtgc agcagatcat
cttcttcctg 1380 ggaaccacgg ccctggcctt cctggtgctc atgcctgtgc
tccatggcag gaacctcctg 1440 ctcttccgtt ccctggagtc ctcgtggccc
ttctggctga ctttggccct ggctgtgatc 1500 ctgcagaaca tggcagccca
ttgggtcttc ctggagactc atgatggaca cccacagctg 1560 accaaccggc
gagtgctcta tgcagccacc tttcttctct tccccctcaa tgtgctggtg 1620
ggtgccatgg tggccacctg gcgagtgctc ctctctgccc tctacaacgc catccacctt
1680 ggccagatgg acctcagcct gctgccaccg agagccgcca ctctcgaccc
cggctactac 1740 acgtaccgaa acttcttgaa gattgaagtc agccagtcgc
atccagccat gacagccttc 1800 tgctccctgc tcctgcaagc gcagagcctc
ctacccagga ccatggcagc cccccaggac 1860 agcctcagac caggggagga
agacgaaggg atgcagctgc tacagacaaa ggactccatg 1920 gccaagggag
ctaggcccgg ggccagccgc ggcagggctc gctggggtct ggcctacacg 1980
ctgctgcaca acccaaccct gcaggtcttc cgcaagacgg ccctgttggg tgccaatggt
2040 gcccagccct gagggcaggg aaggtcaacc cacctgccca tctgtgctga
ggcatgttcc 2100 tgcctaccat cctcctccct ccccggctct cctcccagca
tcacaccagc catgcagcca 2160 gcaggtcctc cggatcactg tggttgggtg
gaggtctgtc tgcactggga gcctcaggag 2220 ggctctgctc cacccacttg
gctatgggag agccagcagg ggttctggag aaaaaaactg 2280 gtgggttagg
gccttggtcc aggagccagt tgagccaggg cagccacatc caggcgtctc 2340
cctaccctgg ctctgccatc agccttgaag ggcctcgatg aagccttctc tggaaccact
2400 ccagcccagc tccacctcag ccttggcctt cacgctgtgg aagcagccaa
ggcacttcct 2460 caccccctca gcgccacgga cctctctggg gagtggccgg
aaagctcccg gtcctctggc 2520 ctgcagggca gcccaagtca tgactcagac
caggtcccac actgagctgc ccacactcga 2580 gagccagata tttttgtagt
ttttatgcct ttggctatta tgaaagaggt tagtgtgttc 2640 cctgcaataa
acttgttcct gagaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2700
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa 2732 2 667 PRT Homo sapiens 2
Met Ser Ser Gln Pro Ala Gly Asn Gln Thr Ser Pro Gly Ala Thr Glu 1 5
10 15 Asp Tyr Ser Tyr Gly Ser Trp Tyr Ile Asp Glu Pro Gln Gly Gly
Glu 20 25 30 Glu Leu Gln Pro Glu Gly Glu Val Pro Ser Cys His Thr
Ser Ile Pro 35 40 45 Pro Gly Leu Tyr His Ala Cys Leu Ala Ser Leu
Ser Ile Leu Val Leu 50 55 60 Leu Leu Leu Ala Met Leu Val Arg Arg
Arg Gln Leu Trp Pro Asp Cys 65 70 75 80 Val Arg Gly Arg Pro Gly Leu
Pro Ser Pro Val Asp Phe Leu Ala Gly 85 90 95 Asp Arg Pro Arg Ala
Val Pro Ala Ala Val Phe Met Val Leu Leu Ser 100 105 110 Ser Leu Cys
Leu Leu Leu Pro Asp Glu Asp Ala Leu Pro Phe Leu Thr 115 120 125 Leu
Ala Ser Ala Pro Ser Gln Asp Gly Lys Thr Glu Ala Pro Arg Gly 130 135
140 Ala Trp Lys Ile Leu Gly Leu Phe Tyr Tyr Ala Ala Leu Tyr Tyr Pro
145 150 155 160 Leu Ala Ala Cys Ala Thr Ala Gly His Thr Ala Ala His
Leu Leu Gly 165 170 175 Ser Thr Leu Ser Trp Ala His Leu Gly Val Gln
Val Trp Gln Arg Ala 180 185 190 Glu Cys Pro Gln Val Pro Lys Ile Tyr
Lys Tyr Tyr Ser Leu Leu Ala 195 200 205 Ser Leu Pro Leu Leu Leu Gly
Leu Gly Phe Leu Ser Leu Trp Tyr Pro 210 215 220 Val Gln Leu Val Arg
Ser Phe Ser Arg Arg Thr Gly Ala Gly Ser Lys 225 230 235 240 Gly Leu
Gln Ser Ser Tyr Ser Glu Glu Tyr Leu Arg Asn Leu Leu Cys 245 250 255
Arg Lys Lys Leu Gly Ser Ser Tyr His Thr Ser Lys His Gly Phe Leu 260
265 270 Ser Trp Ala Arg Val Cys Leu Arg His Cys Ile Tyr Thr Pro Gln
Pro 275 280 285 Gly Phe His Leu Pro Leu Lys Leu Val Leu Ser Ala Thr
Leu Thr Gly 290 295 300 Thr Ala Ile Tyr Gln Val Ala Leu Leu Leu Leu
Val Gly Val Val Pro 305 310 315 320 Thr Ile Gln Lys Val Arg Ala Gly
Val Thr Thr Asp Val Ser Tyr Leu 325 330 335 Leu Ala Gly Phe Gly Ile
Val Leu Ser Glu Asp Lys Gln Glu Val Val 340 345 350 Glu Leu Val Lys
His His Leu Trp Ala Leu Glu Val Cys Tyr Ile Ser 355 360 365 Ala Leu
Val Leu Ser Cys Leu Leu Thr Phe Leu Val Leu Met Arg Ser 370 375 380
Leu Val Thr His Arg Thr Asn Leu Arg Ala Leu His Arg Gly Ala Ala 385
390 395 400 Leu Asp Leu Ser Pro Leu His Arg Ser Pro His Pro Ser Arg
Gln Ala 405 410 415 Ile Phe Cys Trp Met Ser Phe Ser Ala Tyr Gln Thr
Ala Phe Ile Cys 420 425 430 Leu Gly Leu Leu Val Gln Gln Ile Ile Phe
Phe Leu Gly Thr Thr Ala 435 440 445 Leu Ala Phe Leu Val Leu Met Pro
Val Leu His Gly Arg Asn Leu Leu 450 455 460 Leu Phe Arg Ser Leu Glu
Ser Ser Trp Pro Phe Trp Leu Thr Leu Ala 465 470 475 480 Leu Ala Val
Ile Leu Gln Asn Met Ala Ala His Trp Val Phe Leu Glu 485 490 495 Thr
His Asp Gly His Pro Gln Leu Thr Asn Arg Arg Val Leu Tyr Ala 500 505
510 Ala Thr Phe Leu Leu Phe Pro Leu Asn Val Leu Val Gly Ala Met Val
515 520 525 Ala Thr Trp Arg Val Leu Leu Ser Ala Leu Tyr Asn Ala Ile
His Leu 530 535 540 Gly Gln Met Asp Leu Ser Leu Leu Pro Pro Arg Ala
Ala Thr Leu Asp 545 550 555 560 Pro Gly Tyr Tyr Thr Tyr Arg Asn Phe
Leu Lys Ile Glu Val Ser Gln 565 570 575 Ser His Pro Ala Met Thr Ala
Phe Cys Ser Leu Leu Leu Gln Ala Gln 580 585 590 Ser Leu Leu Pro Arg
Thr Met Ala Ala Pro Gln Asp Ser Leu Arg Pro 595 600 605 Gly Glu Glu
Asp Glu Gly Met Gln Leu Leu Gln Thr Lys Asp Ser Met 610 615 620 Ala
Lys Gly Ala Arg Pro Gly Ala Ser Arg Gly Arg Ala Arg Trp Gly 625 630
635 640 Leu Ala Tyr Thr Leu Leu His Asn Pro Thr Leu Gln Val Phe Arg
Lys 645 650 655 Thr Ala Leu Leu Gly Ala Asn Gly Ala Gln Pro 660 665
3 676 DNA Homo sapiens misc_feature (0)...(0) n = A, T, C or G 3
gtgctctccg aggacaagca ggaggnggtg gagctggtga agcaccatct gtgggctctg
60 gaagtgtgct acatctcagc cttggtcttg tcctgcttac tcaccttcct
ggtcctgatg 120 cgctcactgg tgacacacag gaccaacctt cgagctctgc
accgaggagc tgccctggac 180 ttgagtccct tgcatcggag tccccatccc
tcccgccaag ccatattctg ttggatgagc 240 ttcagtgcct accagacagc
ctttatctgc cttgggctcc tggtgcagca gatcatcttc 300 ttcctgggaa
ccacggccct ggccttcctg gtgctcatgc ctgtgctcca tggcaggaac 360
ctcctgctct tccgttccct ggagtcctcg tggcccttct ggctgacttt ggccctggct
420 gtgatcctgc agaacatggc agcccattgg gtcttcctgg agactcatga
tggacaccca 480 cagctgacca accggcgagt gctctatgca gccacctttc
ttctcttccc cctcaatgtg 540 ctggtgggtg ccatggtggc cacctggcga
gtgctcctct ctgccctcta caacgccatc 600 caccttggcc agatggacct
cagcctgctg ccaccgagag ccgccactct cgaccccggc 660 tactacacgt accgaa
676 4 2777 DNA Homo sapiens 4 cacaaccagc cacccctcta ggatcccagc
ccagctggtg ctgggctcag aggagaaggc 60 cccgtgttgg gagcaccctg
cttgcctgga gggacaagtt tccgggagag atcaataaag 120 gaaaggaaag
agacaaggaa gggagaggtc aggagagcgc ttgattggag gagaagggcc 180
agagaatgtc gtcccagcca gcagggaacc agacctcccc cggggccaca gaggactact
240 cctatggcag ctggtacatc gatgagcccc aggggggcga ggagctccag
ccagaggggg 300 aagtgccctc ctgccacacc agcataccac ccggcctgta
ccacgcctgc ctggcctcgc 360 tgtcaatcct tgtgctgctg ctcctggcca
tgctggtgag gcgccgccag ctctggcctg 420 actgtgtgcg tggcaggccc
ggcctgccca ggccccgggc agtgcctgct gctgttttca 480 tggtcctcct
gagctccctg tgtttgctgc tccccgacga ggacgcattg cccttcctga 540
ctctcgcctc agcacccagc caagatggga aaactgaggc tccaagaggg gcctggaaga
600 tactgggact gttctattat gctgccctct actaccctct ggctgcctgt
gccacggctg 660 gccacacagc tgcacacctg ctcggcagca cgctgtcctg
ggcccacctt ggggtccagg 720 tctggcagag ggcagagtgt ccccaggtgc
ccaagatcta caagtactac tccctgctgg 780 cctccctgcc tctcctgctg
ggcctcggat tcctgagcct ttggtaccct gtgcagctgg 840 tgagaagctt
cagccgtagg acaggagcag gctccaaggg gctgcagagc agctactctg 900
aggaatatct gaggaacctc ctttgcagga agaagctggg aagcagctac cacacctcca
960 agcatggctt cctgtcctgg gcccgcgtct gcttgagaca ctgcatctac
actccacagc 1020 caggattcca tctcccgctg aagctggtgc tttcagctac
actgacaggg acggccattt 1080 accaggtggc cctgctgctg ctggtgggcg
tggtacccac tatccagaag gtgagggcag 1140 gggtcaccac ggatgtctcc
tacctgctgg ccggctttgg aatcgtgctc tccgaggaca 1200 agcaggaggt
ggtggagctg gtgaagcacc atctgtgggc tctggaagtg tgctacatct 1260
cagccttggt cttgtcctgc ttactcacct tcctggtcct gatgcgctca ctggtgacac
1320 acaggaccaa ccttcgagct ctgcaccgag gagctgccct ggacttgagt
cccttgcatc 1380 ggagtcccca tccctcccgc caagccatat tctgttggat
gagcttcagt gcctaccaga 1440 cagcctttat ctgccttggg ctcctggtgc
agcagatcat cttcttcctg ggaaccacgg 1500 ccctggcctt cctggtgctc
atgcctgtgc tccatggcag gaacctcctg ctcttccgtt 1560 ccctggagtc
ctcgtggccc ttctggctga ctttggccct ggctgtgatc ctgcagaaca 1620
tggcagccca ttgggtcttc ctggagactc atgatggaca cccacagctg accaaccggc
1680 gagtgctcta tgcagccacc tttcttctct tccccctcaa tgtgctggtg
ggtgccatag 1740 tggccacctg gcgagtgctc ctctctgccc tctacaacgc
catccacctt ggccagatgg 1800 acctcagcct gctgccaccg agagccgcca
ctctcgaccc cggctactac acgtaccgaa 1860 acttcttgaa gattgaagtc
agccagtcgc atccagccat gacagccttc tgctccctgc 1920 tcctgcaagc
gcagagcctc ctacccagga ccatggcagc cccccaggac agcctcagac 1980
caggggagga agacgaaggg atgcagctgc tacagacaaa ggactccatg gccaagggag
2040 ctaggcccgg ggccagccgc ggcagggctc gctggggtct ggcctacacg
ctgctgcaca 2100 acccaaccct gcaggtcttc cgcaagacgg ccctgttggg
tgccaatggt gcccagccct 2160 gagggcaggg aaggtcaacc cacctgccca
tctgtgctga ggcatgttcc tgcctaccac 2220 ctcctccctc cccggctctc
ctcccagcat cacaccagcc atgcagccag caggtcctcc 2280 ggatcactgt
ggttgggtgg aggtctgtct gcactgggag cctcaggagg gctctgctcc 2340
acccacttgg ctatgggaga gccagcaggg gttctggaga aagaaactgg tgggttaggg
2400 ccttggtcca ggagccagtt gagccagggc agccacatcc aggcgtctcc
ctaccctggc 2460 tctgccatca gccttgaagg gcctcgatga agccttctct
ggaaccactc cagcccagct 2520 ccacctcagc cttggccttc acgctgtgga
agcagccaag gcacttcctc accccctcag 2580 cgccacggac ctctctgggg
agtggccgga aagctcccgg gcctctggcc tgcagggcag 2640 cccaagtcat
gactcagacc aggtcccaca ctgagctgcc cacactcgag agccagatat 2700
ttttgtagtt tttatgcctt tggctattat gaaagaggtt agtgtgttcc ctgcaataaa
2760 cttgttcctg agaaaaa 2777 5 658 PRT Homo sapiens 5 Met Ser Ser
Gln Pro Ala Gly Asn Gln Thr Ser Pro Gly Ala Thr Glu 1 5 10 15 Asp
Tyr Ser Tyr Gly Ser Trp Tyr Ile Asp Glu Pro Gln Gly Gly Glu 20 25
30 Glu Leu Gln Pro Glu Gly Glu Val Pro Ser Cys His Thr Ser Ile Pro
35 40 45 Pro Gly Leu Tyr His Ala Cys Leu Ala Ser Leu Ser Ile Leu
Val Leu 50 55 60 Leu Leu Leu Ala Met Leu Val Arg Arg Arg Gln Leu
Trp Pro Asp Cys 65 70 75 80 Val Arg Gly Arg Pro Gly Leu Pro Arg Pro
Arg Ala Val Pro Ala Ala 85 90 95 Val Phe Met Val Leu Leu Ser Ser
Leu Cys Leu Leu Leu Pro Asp Glu 100 105 110 Asp Ala Leu Pro Phe Leu
Thr Leu Ala Ser Ala Pro Ser Gln Asp Gly 115 120 125 Lys Thr Glu Ala
Pro Arg Gly Ala Trp Lys Ile Leu Gly Leu Phe Tyr 130 135 140 Tyr Ala
Ala Leu Tyr Tyr Pro Leu Ala Ala Cys Ala Thr Ala Gly His 145 150 155
160 Thr Ala Ala His Leu Leu Gly Ser Thr Leu Ser Trp Ala His Leu Gly
165 170 175 Val Gln Val Trp Gln Arg Ala Glu Cys Pro Gln Val Pro Lys
Ile Tyr 180 185 190 Lys Tyr Tyr Ser Leu Leu Ala Ser Leu Pro Leu Leu
Leu Gly Leu Gly 195 200 205 Phe Leu Ser Leu Trp Tyr Pro Val Gln Leu
Val Arg Ser Phe Ser Arg 210 215 220 Arg Thr Gly Ala Gly Ser Lys Gly
Leu Gln Ser Ser Tyr Ser Glu Glu 225 230 235 240 Tyr Leu Arg Asn Leu
Leu Cys Arg Lys Lys Leu Gly Ser Ser Tyr His 245 250 255 Thr Ser Lys
His Gly Phe Leu Ser Trp Ala Arg Val Cys Leu Arg His 260 265 270 Cys
Ile Tyr Thr Pro Gln Pro Gly Phe His Leu Pro Leu Lys Leu Val 275 280
285 Leu Ser Ala Thr Leu Thr Gly Thr Ala Ile Tyr Gln Val Ala Leu Leu
290 295 300 Leu Leu Val Gly Val Val Pro Thr Ile Gln Lys Val Arg Ala
Gly Val 305 310 315 320 Thr Thr Asp Val Ser Tyr Leu Leu Ala Gly Phe
Gly Ile Val Leu Ser 325 330 335 Glu Asp Lys Gln Glu Val Val Glu Leu
Val Lys His His Leu Trp Ala 340 345 350 Leu Glu Val Cys Tyr Ile Ser
Ala Leu Val Leu Ser Cys Leu Leu Thr 355 360 365 Phe Leu Val Leu Met
Arg Ser Leu Val Thr His Arg Thr Asn Leu Arg 370 375 380 Ala Leu His
Arg Gly Ala Ala Leu Asp Leu Ser Pro Leu His Arg Ser 385 390 395 400
Pro His Pro Ser Arg Gln Ala Ile Phe Cys Trp Met Ser Phe Ser Ala 405
410 415 Tyr Gln Thr Ala Phe Ile Cys Leu Gly Leu Leu Val Gln Gln Ile
Ile 420 425 430 Phe Phe Leu Gly Thr Thr Ala Leu Ala Phe Leu Val Leu
Met Pro Val 435 440 445 Leu His Gly Arg Asn Leu Leu Leu Phe Arg Ser
Leu Glu Ser Ser Trp 450 455 460 Pro Phe Trp Leu Thr Leu Ala Leu Ala
Val Ile Leu Gln Asn Met Ala 465 470 475 480 Ala His Trp Val Phe Leu
Glu Thr His Asp Gly His Pro Gln Leu Thr 485 490 495 Asn Arg Arg Val
Leu Tyr Ala Ala Thr Phe Leu Leu Phe Pro Leu Asn 500 505 510 Val Leu
Val Gly Ala Ile Val Ala Thr Trp Arg Val Leu Leu Ser Ala 515 520 525
Leu Tyr Asn Ala Ile His Leu Gly Gln Met Asp Leu Ser Leu Leu Pro 530
535 540 Pro Arg Ala Ala Thr Leu Asp Pro Gly Tyr Tyr Thr Tyr Arg Asn
Phe 545 550 555 560 Leu Lys Ile Glu Val Ser Gln Ser His Pro Ala Met
Thr Ala Phe Cys 565 570 575 Ser Leu Leu Leu Gln Ala Gln Ser Leu Leu
Pro Arg Thr Met Ala Ala 580 585 590 Pro Gln Asp Ser Leu Arg Pro Gly
Glu Glu Asp Glu Gly Met Gln Leu 595 600 605 Leu Gln Thr Lys Asp Ser
Met Ala Lys Gly Ala Arg Pro Gly Ala Ser 610 615 620 Arg Gly Arg Ala
Arg Trp Gly Leu Ala Tyr Thr Leu Leu His Asn Pro 625 630 635 640 Thr
Leu Gln Val Phe Arg Lys Thr Ala Leu Leu Gly Ala Asn Gly Ala 645 650
655 Gln Pro 6 23 DNA Artificial Sequence Artificial Sequence =
synthetic oligonucleotide 6 cacactcgag agccagatat ttt 23 7 23 DNA
Artificial Sequence Artificial Sequence = synthetic oligonucleotide
7 aacaagttta ttgcagggaa cac 23 8 36 DNA Artificial Sequence
Artificial Sequence = synthetic oligonucleotide 8 tgtagttttt
atgcctttgg ctattatgaa agaggt 36 9 19 DNA Artificial Sequence
Artificial Sequence = synthetic oligonucleotide 9 agaccaggtc
ccacactga
19 10 24 DNA Artificial Sequence Artificial Sequence = synthetic
oligonucleotide 10 ttcataatag ccaaaggcat aaaa 24 11 23 DNA
Artificial Sequence Artificial Sequence = synthetic oligonucleotide
11 ctgcccacac tcgagagcca gat 23 12 17 PRT Artificial Sequence
Artificial Sequence = synthetic peptide 12 Met Lys His Gln His Gln
His Gln His Gln His Gln His Gln Met His 1 5 10 15 Gln 13 9 PRT Homo
sapiens 13 Ser Pro Val Asp Phe Leu Ala Gly Asp 1 5 14 20 DNA
Artificial Sequence Artificial Sequence = synthetic oligonucleotide
14 gaagatggtg atgggatttc 20 15 19 DNA Artificial Sequence
Artificial Sequence = synthetic oligonucleotide 15 gaaggtgaag
gtcggagtc 19 16 20 DNA Artificial Sequence Artificial Sequence =
synthetic oligonucleotide 16 caagcttccc gttctcagcc 20 17 19 DNA
Artificial Sequence Artificial Sequence = synthetic oligonucleotide
17 tgcaccacca actgcttag 19 18 19 DNA Artificial Sequence Artificial
Sequence = synthetic oligonucleotide 18 ggatgcaggg atgatgttc 19 19
23 DNA Artificial Sequence Artificial Sequence = synthetic
oligonucleotide 19 cagaagactg tggatggccc ctc 23 20 20 DNA
Artificial Sequence Artificial Sequence = synthetic oligonucleotide
20 gtaggccagg gctatttctg 20 21 20 DNA Artificial Sequence
Artificial Sequence = synthetic oligonucleotide 21 tgtctcatga
gggtccaaaa 20 22 21 DNA Artificial Sequence Artificial Sequence =
synthetic oligonucleotide 22 tggcgctctt ctctcctcgg t 21 23 19 DNA
Artificial Sequence Artificial Sequence = synthetic oligonucleotide
23 tcgccaagct actggctaa 19 24 18 DNA Artificial Sequence Artificial
Sequence = synthetic oligonucleotide 24 cttggctgtg ccagttca 18 25
28 DNA Artificial Sequence Artificial Sequence = synthetic
oligonucleotide 25 aaccaagcat cgttgtgcaa tacaactc 28 26 21 DNA
Artificial Sequence Artificial Sequence = synthetic oligonucleotide
26 gccagtacag gatctggaaa g 21 27 26 DNA Artificial Sequence
Artificial Sequence = synthetic oligonucleotide 27 catatctcat
cagagagcat ctaaaa 26 28 22 DNA Artificial Sequence Artificial
Sequence = synthetic oligonucleotide 28 aagcttttag cctgcccagc ca
22
* * * * *
References